JP2006296350A - Method for producing nucleic acid construct in which a plurality of nucleic acids are linked and cloning of nucleic acid using the method - Google Patents

Abstract

【Task】
A nucleic acid construct is produced by linking a plurality of nucleic acid molecules without using a restriction enzyme.
[Solution]
The present invention includes the following: (a) a step of amplifying a first nucleic acid molecule to obtain a first double-stranded amplification product; (b) a second nucleic acid molecule being amplified to obtain a second double-stranded amplification product Wherein the 3 ′ end portion of the first double-stranded amplification product and the 5 ′ end portion of the second double-stranded amplification product are hybridized only with a specific sequence. (C) mixing the first and second double-stranded amplification products obtained by steps (a) and (b), and A step of denatured into a strand and hybridizing under a condition in which only the specific sequence hybridizes; and (d) a nucleic acid amplification reaction using the mixture of (c) as a template to obtain a linked nucleic acid amplification product. A method comprising the steps is provided.
[Selection] Figure 2

Description

  The present invention relates to a nucleic acid construct obtained by linking a plurality of desired nucleic acid molecules and a method for producing the same. More specifically, the present invention relates to a method of linking a plurality of desired nucleic acids through specific hybridization of nucleic acids and nucleic acid synthesis without using restriction enzymes.

  In the fields of molecular biology, tissue and organ regeneration, regenerative medicine and regenerative medicine, gene therapy, diagnostics, and drug development, a technique for producing an arbitrary nucleic acid construct is important. The production of nucleic acid constructs is also important for the cloning of any gene.

  Conventionally, a gene construct from a plurality of nucleic acid molecules is produced by a method using a restriction enzyme, which is a sequence-specific nuclease that recognizes a specific sequence of DNA. In this technique, a restriction enzyme that cuts a desired site of a nucleic acid molecule but does not cut other sites of the nucleic acid molecule is selected, and a nucleic acid molecule fragment having the same cut surface or a smoothed surface is converted into DNA. This is accomplished by ligation with ligase (see, eg, FIG. 1).

  In order to use a restriction enzyme, it is necessary to create a distribution map (ie, restriction enzyme map) of cleavage sites by restriction enzymes peculiar to the target nucleic acid sequence, and knowledge or software for that is required. Referring to FIG. 1, amplification of gene fragment A and gene fragment B by a general PCR method and a method for constructing a new gene construct A + B are shown. Here, it is essential that a restriction enzyme cleavage site exists at an appropriate position in the nucleic acid molecule, but there are many cases where an appropriate restriction enzyme cleavage site does not exist at an appropriate position of the nucleic acid desired to be linked. In such a case, it is necessary to artificially insert a nucleic acid portion having an appropriate cleavage site. In addition, in order to use a restriction enzyme, it is necessary to have a knowledge of the distribution map (ie, restriction enzyme map) of the cleavage site by the restriction enzyme unique to the gene.

  Nucleic acid production, including artificial insertion of nucleic acid moieties, is often performed using the polymerase chain reaction (PCR) method (Saiki RK, Scharf S., Falona F., Mullis KB, Horn GT, Erlich). HA, Arnheim N., (1985) Science 230 (4732): 1350-1354 (Non-Patent Document 1); Mullis K., Fallona F., “Methods in Enzymology (Edited by Wu. R.) Academic. Press, San Diego "(1987), 155 (Non-Patent Document 2); Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., Errich, H., "Cold Spring Harbo Symposia on Quantiative biology, 101 ", performed by the method using a (1986), 263 pp ～273 (Non-Patent Document 3)). In this method, a primer having a form in which an adapter (or linker) sequence including a restriction enzyme recognition sequence is added to the 5 ′ end is prepared, and PCR is carried out using this primer to obtain a desired insertion portion at the end portion. An amplification product, which is a nucleic acid molecule comprising, can be obtained.

  However, such an operation for using a restriction enzyme requires a very complicated process and takes time. Therefore, it has been necessary to develop a technique for producing a nucleic acid construct by linking a plurality of nucleic acid molecules without using a restriction enzyme.

  As a known method for linking many nucleic acid fragments without using a restriction enzyme, there is a method described in International Publication No. 99/16904 (Patent Document 1) that combines multiplex PCR and linking PCR. . In this method, a primer comprising a portion complementary to the 3 ′ end portion of the sense strand of the first nucleic acid and a short tail portion present on the 3 ′ side thereof, and a second tail Multiplex PCR using a primer containing a portion complementary to the 5 ′ end portion of the antisense strand of the nucleic acid and a short tail portion present on the 5 ′ side thereof is followed by PCR using this amplification product as a template. Thus, the desired nucleic acid is linked. This method has the advantage that many nucleic acid fragments can be prepared by one PCR and the ligation step can be performed together, but an unnecessary sequence included in the tail portion is included in the final nucleic acid fragment. There are drawbacks.

  As another known method, there is a method of ligating DNA using a DNA polymerase having an endogenous exonuclease activity, which is described in International Publication No. 01/066675 (Patent Document 2). In this method, DNA fragments having overlapping portions with each other between adjacent nucleic acids for which ligation is desired are prepared and ligated using the endogenous exonuclease activity of DNA polymerase.

  WO 98/04746 (Patent Document 3) discloses a nucleic acid branch amplification method. However, in this method, the use of paramagnetic beads is indispensable, and the process is very complicated.

  JP-A-8-268 (Patent Document 4) discloses a method for producing linked double-stranded DNA. Here, it is assumed that a sticky terminal sequence is used, and the process is very complicated.

  JP2003-189890 (Patent Document 5) describes a method for producing a linear DNA fragment. However, a method for constructing a plurality of linked nucleic acids is not described. In Patent Document 5, it is pointed out that only the content of preparing a template for protein expression is used, and a PCR reaction that may impair accuracy is used every time, so that accuracy is applied.

  WO 00/66768 describes a method for amplifying nucleic acids. However, a method for constructing a plurality of linked nucleic acids is not described.

  As described above, in the conventional method, even if the linked nucleic acid cannot be amplified or not, a very complicated process is required and time is required.

  From such a background, the advent of a technique for creating an arbitrary gene construct without using a restriction enzyme and a gene cloning technique are awaited.

  In addition, it is pointed out that PCR required for cloning does not contain 0% errors in reading the sequence.

  In the post-genome era, accurate base sequence information is important in analyzing various genes (full-length sequences) and gene domains (partial sequences). A single base mistake (which may also affect the amino acid sequence) is an important problem, and there are many genes that actually become dysfunctional due to a single base difference in mutation analysis or the like.

  It is not preferable to use a linked nucleic acid construct obtained by PCR as a probe and a protein expression template as it is for an accurate functional analysis (for example, Patent Document 5), and it is used for gene analysis in the post-genomic era. It is essential that the nucleic acid constructs have accurate sequence information. Therefore, it is desirable not to include inaccurate information resulting from PCR or the like as much as possible.

If the target nucleic acid construct is cloned into a vector such as a plasmid, the accuracy of the sequence information of the underlying nucleic acid construct can be confirmed by examining the clone, and a nucleic acid construct having the same sequence (and a probe or protein using the same) Expression) is always available. From the above, it is important that the linked nucleic acid construct can be cloned. Therefore, the advent of such a technology is awaited.
International Publication No. 99/16904 Pamphlet WO 01/066675 Pamphlet International Publication No. 98/04746 JP-A-8-268 JP 2003-189890 A International Publication No. 00/66768 Saiki R.D. K. , Scharf S .; Faloona F. Mullis K .; B. Horn G. T. T. et al. Erlich H., et al. A. Arnheim N .; (1985) Science 230 (4732): 1350-1354. Mullis K.M. Faloona F. "Methods in Enzymology (ed. By Wu. R.) Academic Press, San Diego" (1987), p.155. Mullis, K.M. Faloona, F .; Scharf, S .; Saiki, R .; Horn, G .; Erlich, H .; , "Cold Spring Harbor Symposia on Quantitative Biology, 101" (1986), pp. 263-273.

  In view of the above, an object is to provide a technique for preparing a nucleic acid construct by linking a plurality of nucleic acid molecules without using restriction enzymes, simply, rapidly, accurately and cost-effectively.

  The object of the present invention is to solve the following problems. That is, without using restriction enzymes, any two kinds of gene fragments can be joined together to produce a nucleic acid construct, gene cloning can be performed without using restriction enzymes, and PCR methods using conventional restriction enzymes in combination. The objective is to provide a much simpler and faster method compared to the method used, and to provide a method corresponding to the creation and cloning of all types of gene constructs.

  The present invention provides a nucleic acid construct in which a plurality of sequences are linked without using a restriction enzyme by partially using a hybridizing sequence as part of two sets of primer pairs in a nucleic acid amplification reaction. Solve the above problems by success. The present invention also provides a method for performing gene cloning.

In the present invention, simple cloning has been achieved. It can be said that the inclusion of a vector in the ligated nucleic acid construct does not require any special work and can be easily cloned by a molecular biological technique. Therefore, the present invention provides the following:
(1) A method for producing a nucleic acid construct in which a plurality of nucleic acid molecules are linked, wherein the method is as follows:
(A) amplifying the first nucleic acid molecule to obtain a first double-stranded amplification product;
(B) a step of amplifying a second nucleic acid molecule to obtain a second double-stranded amplification product, wherein the 3′-end portion of the first double-stranded amplification product and the second double-stranded product Amplifying the 5 ′ end portion of the amplification product to have a sequence that hybridizes under conditions in which only specific sequences hybridize;
(C) A condition in which the first and second double-stranded amplification products obtained in steps (a) and (b) are mixed, denatured into single strands, and only the specific sequence hybridizes. And (d) performing a nucleic acid amplification reaction using the mixture of (c) as a template to obtain a linked nucleic acid amplification product.
(2) The method according to item 1, wherein the method is as follows:
(A) a first reverse single-stranded nucleic acid molecule capable of hybridizing under the nucleic acid amplification conditions to the 3 ′ end portion of the first nucleic acid molecule using the first nucleic acid molecule as a template, and the first nucleic acid molecule Using the first forward single-stranded nucleic acid molecule hybridizable under the above-mentioned nucleic acid amplification conditions as a primer to the 5 ′ end portion of the complementary strand, a nucleic acid amplification reaction is performed to obtain a first double-stranded amplification product Process;
(B) a second reverse single-stranded nucleic acid molecule capable of hybridizing to the 3 ′ end portion of the second nucleic acid molecule under the nucleic acid amplification conditions using the second nucleic acid molecule as a template, and the second nucleic acid Using the second forward single-stranded nucleic acid molecule hybridizable under the above-mentioned nucleic acid amplification conditions as a primer to the 5 ′ end portion of the complementary strand of the molecule, a nucleic acid amplification reaction is carried out to obtain a second double-stranded amplification product Wherein the first forward single stranded nucleic acid molecule and the second reverse single stranded nucleic acid molecule comprise a 3 ′ end portion of the first double stranded amplification product, A step wherein the 5 ′ end portion of the second double-stranded amplification product is designed to hybridize under conditions in which only specific sequences hybridize;
(C) The first and second double-stranded amplification products obtained in steps (a) and (b) are mixed, denatured into single strands, and under conditions where only specific sequences hybridize. (D) using the mixture of (c) as a template, and using the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule as primers. Performing a nucleic acid amplification reaction to obtain a linked nucleic acid amplification product.
(3) The method according to item 1, wherein:
(E) amplifying the double-stranded nucleic acid molecule obtained by step (d),
Further comprising a method.
(4) The reverse single-stranded nucleic acid molecule and the forward single-stranded nucleic acid molecule are sufficient to specifically hybridize and have sequences and lengths that do not interfere with the amplification reaction. 2. The method according to 2.
(5) the first forward single-stranded nucleic acid molecule has a sequence that hybridizes with the 5 ′ end portion of the second nucleic acid molecule under the condition that only the specific sequence hybridizes; or The second reverse single-stranded nucleic acid molecule has a sequence that hybridizes with the 3 ′ end portion of the first nucleic acid molecule under the condition that only the specific sequence hybridizes, or both. The method according to item 2, characterized in that:
(6) The first forward single-stranded nucleic acid molecule has a sequence complementary to the 5 ′ end portion of the second nucleic acid molecule, or the second reverse single-stranded nucleic acid molecule is the above 6. The method according to item 5, characterized in that it has a sequence complementary to the 3 ′ terminal portion of the first nucleic acid molecule, or both.
(7) The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are hybridized only with the specific sequence in both the first nucleic acid molecule and the second nucleic acid molecule. Item 3. The method according to Item 2, comprising a tag sequence that does not hybridize under the conditions of the tag and a tag complementary sequence that hybridizes under the condition that only the specific sequence hybridizes to the tag sequence.
(8) The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are hybridized only with the specific sequence in both the first nucleic acid molecule and the second nucleic acid molecule. Item 8. The method according to Item 7, comprising a tag sequence that does not hybridize under the conditions of the tag and the tag complementary sequence that is complementary to the tag sequence.
(9) The method according to item 1, wherein at least one of the first nucleic acid molecule or the second nucleic acid molecule has at least a part or all of the sequence of the vector.
(10) The method according to item 9, wherein the vector is a cloning vector.
(11) Amplification of the nucleic acid includes polymerase chain reaction (PCR), ligase chain reaction (LCR), gap LCR, strand displacement amplification (SDA), Qβ replicase amplification, transcription amplification system (TAS), self-supporting From a self-sustained sequence replication system (3SR); nucleic acid sequence-based amplification (NASBA); transcription-mediated amplification (TMA), chimeric primer-initiated nucleic acid amplification (ICAN) and loop-mediated isothermal amplification (LAMP) under isothermal conditions The method according to item 1, which is performed by an amplification method selected from the group consisting of:
(12) The method according to item 1, wherein the nucleic acid comprises DNA.
(13) A nucleic acid construct obtained by the method according to item 1.
(14) Two nucleic acid primer sets, the set comprising: a first forward single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule to obtain a first double-stranded amplification product; A first forward single stranded nucleic acid for amplifying the second nucleic acid molecule to obtain a second double stranded amplification product, comprising a first primer set comprising a first reverse single stranded nucleic acid molecule A second primer set comprising a molecule and a second reverse single stranded nucleic acid molecule;
The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule include a 3 ′ end portion of the first double-stranded amplification product and the second double-stranded amplification product. Is designed to hybridize under conditions in which only specific sequences hybridize,
Two nucleic acid primer sets.
(15) A method for designing two nucleic acid primer sets,
A) The inside in which the first nucleic acid molecule and the second nucleic acid molecule are hybridized under the condition that only the specific sequence hybridizes by comparing the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule. Determining whether they have hybridizing sequences to each other;
B) a first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule, and the second nucleic acid molecule Designing a second primer set comprising a second forward single stranded nucleic acid molecule and a second reverse single stranded nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are respectively converted into the first reverse sequence. Primers are designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence is present, the internal hybridizing hybridizes under the condition that only the specific sequence hybridizes to the internal hybridizing sequence and the internal hybridizing sequence. A complementary sequence, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. Designing a primer to include a process,
Including the method.
(16) A method for producing two nucleic acid primer sets,
A) The inside in which the first nucleic acid molecule and the second nucleic acid molecule are hybridized under the condition that only the specific sequence hybridizes by comparing the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule. Determining whether they have hybridizing sequences to each other;
B) a first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule, and the second nucleic acid molecule Designing a second primer set comprising a second forward single stranded nucleic acid molecule and a second reverse single stranded nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are respectively converted into the first reverse sequence. Primers are designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence is present, the internal hybridizing hybridizes under the condition that only the specific sequence hybridizes to the internal hybridizing sequence and the internal hybridizing sequence. A complementary sequence, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. Designing a primer to include; and C) producing a nucleic acid molecule having the sequence of the designed primer.
(17) A program for designing two nucleic acid primer sets,
A) Conditions for allowing the nucleic acid sequence determination means to compare the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule so that only the specific sequences of the first nucleic acid molecule and the second nucleic acid molecule hybridize. Determining whether they have internal hybridizing sequences that hybridize under each other;
B) a first primer set including a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule in the primer designing means; A procedure for designing a second primer set comprising a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule for amplifying a second nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are changed to the first reverse sequence, respectively. Primers are designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule,
In the procedure A), when it is determined that the hybridizing sequence is present, the internal hybridizing hybridizes under the condition that only the specific sequence hybridizes to the internal hybridizing sequence and the internal hybridizing sequence. A complementary sequence, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. Let the primers be designed to include procedures,
Including the program.
(18) A recording medium storing a program for designing two nucleic acid primer sets, wherein the program is
A) Conditions for allowing the nucleic acid sequence determination means to compare the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule so that only the specific sequences of the first nucleic acid molecule and the second nucleic acid molecule hybridize. Determining whether they have internal hybridizing sequences that hybridize under each other;
B) a first primer set including a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule in the primer designing means; A procedure for designing a second primer set comprising a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule for amplifying a second nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are changed to the first reverse sequence, respectively. Primers are designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule,
In the procedure A), when it is determined that the hybridizing sequence is present, the internal hybridizing hybridizes under the condition that only the specific sequence hybridizes to the internal hybridizing sequence and the internal hybridizing sequence. A complementary sequence, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. Let the primers be designed to include procedures,
Including a recording medium.
(19) A device for designing two nucleic acid primer sets,
A) The inside in which the first nucleic acid molecule and the second nucleic acid molecule are hybridized under the condition that only the specific sequence hybridizes by comparing the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule. Nucleic acid sequence determination means for determining whether or not to have hybridizing sequences with each other;
B) a first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule, and the second nucleic acid molecule A primer design means for designing a second primer set comprising a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are changed to the first reverse sequence, respectively. Primers are designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule,
In the procedure A), when it is determined that the hybridizing sequence is present, the internal hybridizing hybridizes under the condition that only the specific sequence hybridizes to the internal hybridizing sequence and the internal hybridizing sequence. A complementary sequence, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. A design apparatus comprising primer design means for designing a primer to include.
(20) A device for synthesizing two nucleic acid primer sets,
A) The inside in which the first nucleic acid molecule and the second nucleic acid molecule are hybridized under the condition that only the specific sequence hybridizes by comparing the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule. Nucleic acid sequence determination means for determining whether or not to have hybridizing sequences with each other;
B) a first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule, and the second nucleic acid molecule A primer design means for designing a second primer set comprising a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are changed to the first reverse sequence, respectively. Primers are designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule,
In the procedure A), when it is determined that the hybridizing sequence is present, the internal hybridizing hybridizes under the condition that only the specific sequence hybridizes to the internal hybridizing sequence and the internal hybridizing sequence. A complementary sequence, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. A synthesis device comprising: a primer design means for designing a primer to include; and C) a means for producing a nucleic acid molecule having the sequence of the designed primer.
(21) A kit for producing a nucleic acid construct in which a plurality of nucleic acid molecules are linked,
A) Two nucleic acid primer sets, the set comprising a first forward single-stranded nucleic acid molecule and a first nucleic acid molecule for amplifying the first nucleic acid molecule to obtain a first double-stranded amplification product. A first forward single-stranded nucleic acid molecule for amplifying the second nucleic acid molecule to obtain a second double-stranded amplification product by comprising a first primer set comprising one reverse single-stranded nucleic acid molecule And a second primer set comprising a second reverse single stranded nucleic acid molecule,
The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule include a 3 ′ end portion of the first double-stranded amplification product and the second double-stranded amplification product. Is designed to hybridize under conditions in which only specific sequences hybridize,
A kit comprising two nucleic acid primer sets; and B) a reaction reagent for an amplification reaction using the primer set.
(22) A kit for cloning a desired nucleic acid molecule,
A) Two nucleic acid primer sets, the set comprising a first forward single-stranded nucleic acid molecule and a first nucleic acid molecule for amplifying the first nucleic acid molecule to obtain a first double-stranded amplification product. A first forward single-stranded nucleic acid molecule for amplifying the second nucleic acid molecule to obtain a second double-stranded amplification product by comprising a first primer set comprising one reverse single-stranded nucleic acid molecule And a second primer set comprising a second reverse single stranded nucleic acid molecule,
The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule include a 3 ′ end portion of the first double-stranded amplification product and the second double-stranded amplification product. Is designed to hybridize under conditions in which only specific sequences hybridize,
Two nucleic acid primer sets; and B) the first nucleic acid molecule as a cloning vector,
Wherein the second nucleic acid molecule is the desired nucleic acid molecule.
(23) A method for cloning a desired nucleic acid molecule using a first nucleic acid molecule that is a cloning vector and a second nucleic acid molecule that is a cloning target, wherein the first nucleic acid A nucleic acid construct in which a plurality of nucleic acid molecules including a molecule and the second nucleic acid molecule are linked is prepared, and the method includes the following:
(A) amplifying the first nucleic acid molecule to obtain a first double-stranded amplification product;
(B) a step of amplifying the second nucleic acid molecule to obtain a second double-stranded amplification product, the 3 ′ end portion of the first double-stranded amplification product, and the second two-stranded amplification product A step wherein the 5 ′ end portion of the strand amplification product is amplified to have a sequence that hybridizes under conditions in which only specific sequences hybridize;
(C) A condition in which the first and second double-stranded amplification products obtained in steps (a) and (b) are mixed, denatured into single strands, and only the specific sequence hybridizes. Hybridizing with:
(D) A method comprising performing a nucleic acid amplification reaction using the mixture of (c) as a template to obtain a linked nucleic acid amplification product in which the desired nucleic acid is cloned.
(24) The method according to item 23, wherein the method is as follows:
(A) using the first nucleic acid molecule as a template, a first reverse single-stranded nucleic acid molecule capable of hybridizing to the 3 ′ end portion of the first nucleic acid molecule under nucleic acid amplification conditions, and the first Using the first forward single-stranded nucleic acid molecule hybridizable under the above-mentioned nucleic acid amplification conditions as a primer to the 5 ′ end portion of the complementary strand of the nucleic acid molecule, a nucleic acid amplification reaction is carried out to obtain a first double-stranded amplification product Obtaining
(B) using the second nucleic acid molecule as a template, a second reverse single-stranded nucleic acid molecule capable of hybridizing to the 3 ′ end portion of the second nucleic acid molecule under the nucleic acid amplification conditions, and the second A second double-stranded amplification is performed by performing a nucleic acid amplification reaction using the second forward single-stranded nucleic acid molecule hybridizable under the above-mentioned nucleic acid amplification conditions as a primer to the 5 ′ end portion of the complementary strand of the nucleic acid molecule Obtaining a product, wherein the first forward single stranded nucleic acid molecule and the second reverse single stranded nucleic acid molecule are the 3 ′ end portion of the first double stranded amplification product, And the 5 ′ end portion of the second double-stranded amplification product are designed to hybridize under conditions where only specific sequences hybridize;
(C) The first and second double-stranded amplification products obtained in steps (a) and (b) are mixed, denatured into single strands, and under conditions where only specific sequences hybridize. (D) using the mixture of (c) as a template, and using the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule as primers. A method comprising a step of performing a nucleic acid amplification reaction to obtain a linked nucleic acid amplification product in which the desired nucleic acid is cloned.
(25) The method according to item 23, wherein:
(E) identifying the desired nucleic acid by sequencing the ligated nucleic acid amplification product obtained in step (d),
Further comprising a method.
(26) The method according to item 23, comprising a step of ligating the ligated nucleic acid amplification product obtained by the step (d) into a circular shape.
(27) The method according to item 26, wherein the step of circularizing comprises phosphorylating the end of the ligated nucleic acid amplification product and ligating with ligase.
(28) The method according to item 26, further comprising the step of introducing the circularized linked nucleic acid amplification product into a host cell and amplifying the linked nucleic acid amplification product.
(29) The item wherein the reverse single-stranded nucleic acid molecule and the forward single-stranded nucleic acid molecule are sufficient to specifically hybridize and have a sequence and length that do not interfere with the amplification reaction. 24. The method according to 24.
(30) the first forward single-stranded nucleic acid molecule has a sequence that hybridizes with the 5 ′ end portion of the second nucleic acid molecule under the condition that only the specific sequence hybridizes; or The second reverse single-stranded nucleic acid molecule has a sequence that hybridizes with the 3 ′ end portion of the first nucleic acid molecule under the condition that only the specific sequence hybridizes, or both. 25. A method according to item 24, characterized in that
(31) The first forward single-stranded nucleic acid molecule has a sequence complementary to the 5 ′ end portion of the second nucleic acid molecule, or the second reverse single-stranded nucleic acid molecule is the above 31. A method according to item 30, characterized in that it has a sequence complementary to the 3 ′ terminal portion of the first nucleic acid molecule, or both.
(32) The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are hybridized only with the specific sequence in both the first nucleic acid molecule and the second nucleic acid molecule. 25. The method according to item 24, comprising: a tag sequence that does not hybridize under the conditions of the tag, and a tag complementary sequence that hybridizes under the condition that only the specific sequence hybridizes to the tag sequence.
(33) The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are hybridized only with the specific sequence in both the first nucleic acid molecule and the second nucleic acid molecule. 33. The method according to item 32, comprising a tag sequence that does not hybridize under the conditions of the tag sequence and the tag complementary sequence that is complementary to the tag sequence.
(34) A cell comprising a nucleic acid construct obtained by the method according to item 1.
(35) A polypeptide encoded by a nucleic acid construct obtained by the method according to item 1.

  Accordingly, it is understood that these and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description with reference to the accompanying drawings.

  By the method according to the present invention, a construct in which two or more nucleic acid molecules are linked can be prepared without using a restriction enzyme. By this method, a nucleic acid construct can be prepared more easily and quickly with a nucleic acid than conventional methods, and gene cloning techniques can be improved.

  The method provided by the present invention is simpler, faster, more accurate and more cost effective than conventional methods and can be applied to any nucleic acid. According to the method of the present invention, it is possible to create a new gene construct by joining any two or more nucleic acid molecules without using a restriction enzyme. In addition, it has become possible to clone any gene without using a restriction enzyme. Compared to a conventional method using an amplification reaction such as a PCR method using a restriction enzyme, a method that is much simpler and quicker was provided for the first time.

  As described above, in Patent Document 1, an unnecessary sequence is included in the final nucleic acid fragment. No means for overcoming this is described or suggested in Patent Document 1. In order to solve this problem, the present invention has found and solved that a complementary primer is prepared with a necessary sequence. Therefore, a method for efficiently performing cloning using this theory was also provided.

  As described above, in Patent Document 2, a restriction enzyme is used. However, this is not applicable when the target nucleic acid contains an endogenous restriction enzyme sequence. In this respect, it can be said that the present case which does not depend on restriction enzymes is excellent.

  As described above, Patent Document 3 has a complicated process. Also, the emphasis is mainly on the efficient amplification of target nucleic acids and their detection, and it does not encompass the creation and cloning of new nucleic acid constructs as shown in this case.

  As described above, Patent Document 4 has a complicated process. This process is simplified by the present invention.

  As described above, Patent Document 5 is not limited to the content of preparing a template for protein expression, but rather than performing the template preparation of a nucleic acid construct using a PCR reaction that may impair accuracy each time. As in the invention, it is cheaper and simpler to use a nucleic acid construct cloned into a vector.

  Patent Document 6 describes a method for nucleic acid amplification, but a plurality of linked nucleic acids cannot be constructed, and the present invention is notable for providing the method.

  The invention will now be described by way of example, with reference to the accompanying drawings, where appropriate. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Therefore, it should be understood that the expression of the singular includes the concept of the plural unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(the term)
As used herein, the terms “polynucleotide”, “oligonucleotide”, “nucleic acid molecule” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives” as long as they do not inhibit the amplification reaction.

  In the present specification, the term “oligonucleotide derivative” or “polynucleotide derivative” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or has an unusual linkage between nucleotides, and is used interchangeably. . Specific examples of such an oligonucleotide include, for example, 2′-O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide. Is an N3′-P5 ′ phosphoramidate bond derivative oligonucleotide, a ribose and phosphodiester bond in the oligonucleotide converted to a peptide nucleic acid bond, uracil in the oligonucleotide is C− A derivative oligonucleotide substituted with 5-propynyluracil, a derivative oligonucleotide where uracil in the oligonucleotide is substituted with C-5 thiazole uracil, and cytosine in the oligonucleotide is C-5 propynylcytosine Substituted derivative oligonucleotides, derivative oligonucleotides in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, derivative oligonucleotides in which the ribose in the DNA is replaced with 2'-O-propylribose Examples thereof include derivative oligonucleotides in which ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-). 98 (1994)). Genes encoding proteins and the like usually take this polynucleotide form.

  The term “nucleotide” as used herein refers to a monomeric unit of DNA consisting of a sugar moiety (pentose sugar), a phosphate group and a nitrogen-containing heterocyclic base. The base is linked to the sugar moiety via a glycoside carbon (1 'carbon of the pentose sugar), and this base-sugar combination is a nucleoside. If the nucleoside contains a phosphate group attached to the 3 'or 5' position of a pentose sugar, this is called a nucleotide. Functionally linked nucleotide sequences are generally referred to herein as “nucleotide sequences” and their grammatical equivalents, with the left-to-right orientation in the normal orientation from the 5 ′ end to the 3 ′ end. It is expressed by a certain rule.

  In the present specification, the “nucleotide” may be natural or non-natural. “Nucleotide derivative” or “nucleotide analog” refers to a substance that is different from a naturally occurring nucleotide but has a function similar to that of the original nucleotide. Such nucleotide derivatives and nucleotide analogs are well known in the art. Examples of such nucleotide derivatives and nucleotide analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-O-methylribonucleotide, peptide-nucleic acid (PNA). .

  When used in an extension reaction, the nucleotide can take the form of a triphosphate. Nucleoside triphosphates include deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), deoxyuridine, which are DNA components. Examples thereof include, but are not limited to, triphosphate (dUTP) and deoxyinosine triphosphate (dITP). In the extension reaction, nucleotides for stopping the reaction include dideoxyadenosine triphosphate (ddATP), dideoxyguanosine triphosphate (ddGTP), dideoxycytidine triphosphate (ddCTP), as used in the dideoxy chain terminator method, Examples include, but are not limited to, dideoxythymidine triphosphate (ddTTP), dideoxyuridine triphosphate (ddUTP), dideoxyinosine triphosphate (ddITP), and the like. Unless otherwise stated, it is understood that “nucleotide” and “nucleotide triphosphate” are used interchangeably in the present invention.

  As used herein, a nucleotide can be “labeled”. Examples of such a label include, but are not limited to, any label using fluorescence, radioactivity, phosphorescence, biotin, DIG, enzyme, and chemiluminescence.

  In this specification, the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others. Examples of such a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like.

  As used herein, “fragment” or “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). . The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute and have the same function (for example, mouse IAP). As long as it has a function such as the above, the above-mentioned number as the upper limit or adjustment is intended to include a number above and below that number (or, for example, 10% above and below). In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.

  As used herein, “nucleotide” refers to a nucleoside in which the sugar moiety is a phosphate ester, and includes DNA, RNA, etc., and may be natural or non-natural. Here, nucleoside refers to a compound in which a base and a sugar form an N-glycoside bond. “Derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). . DNA includes cDNA, genomic DNA, and synthetic DNA.

  As used herein, the term “nucleic acid molecule” includes, but is not limited to, single stranded DNA, double stranded DNA, single stranded RNA.

  As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also encompass one assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is included. The gene product of a protein usually takes the form of a polypeptide, but may be a variant of the polypeptide as long as it has a similar function. Polypeptides having a specific amino acid sequence include fragments, homologues, derivatives and variants thereof.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by a generally recognized one letter code.

The character codes are as follows.
3 letter code of amino acid 1 letter code Meaning Ala A alanine Cys C cysteine Asp D aspartic acid Glu E glutamic acid Phe F phenylalanine Gly G glycine His H histidine Ile I isoleucine Lys K lysine Leu L leucine Met M methionine Asl G Glutamine Arg R Arginine Ser S Serine Thr T Threonine Val V Valine Trp W Tryptophan Tyr Y Tyrosine Asx Asparagine or Aspartate Glx Glutamine or Glutamate Xaa Unknown or other amino acids.
Base symbol Meaning
a adenine g guanine c cytosine t thymine u uracil r guanine or adenine purine y thymine / uracil or cytosine pyrimidine m adenine or cytosine amino group k guanine or thymine / uracil keto group s guanine or cytosine w adenine or thymine / uracil b guanine or cytosine Thymine / uracil d adenine or guanine or thymine / uracil h adenine or cytosine or thymine / uracil v adenine or guanine or cytosine n adenine or guanine or cytosine or thymine / uracil, unknown or other base.

  In the present specification, comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using BLAST, which is a tool for sequence analysis. The identity search can be performed, for example, using NCBI's BLAST 2.2.9 (issued 2004.5.12). In the present specification, the identity value usually refers to a value when the above BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.

  As used herein, “nucleic acid construct” refers to a construct comprising a nucleic acid molecule (eg, DNA, RNA). Usually, a nucleic acid molecule encodes a gene, but is not limited thereto. In addition to the nucleic acid sequence encoding the gene, the nucleic acid construct includes a nucleic acid sequence that includes a control sequence (for example, a promoter) operably linked as necessary (that is, the expression of the nucleic acid can be controlled). As well as, optionally, regulatory sequences (eg, promoters) and heterologous genes operably linked thereto (ie, in frame). It is also within the scope of the present invention to use this construct in combination with other regulatory elements as required.

  As used herein, “amplified product” refers to a product obtained by a nucleic acid amplification reaction, and is usually a nucleic acid molecule. An amplification product obtained by amplifying a nucleic acid molecule contains a sequence substantially identical to the nucleic acid molecule. The amplification product is often double-stranded, and in the present specification, the double-stranded form is also referred to as a double-stranded amplification product. In addition, when referring to an amplification product in which two or more nucleic acid molecules are linked, they are also referred to as “linked nucleic acid amplification products”.

  As used herein, “tag sequence” refers to a sequence that is substantially different from a target nucleic acid molecule (as a template) and that is incorporated into a primer. By using a tag sequence, the present invention has achieved facilitating ligation.

  As used herein, “specific”, when used with a nucleic acid sequence, interacts (eg, hybridizes) with a reference standard nucleic acid sequence at a higher level than the general nucleic acid sequence. That means. There can be one or more specific sequences. Further, when a specific sequence is present, a sequence with higher specificity may be present. Preferably a specific sequence refers to interacting specifically only at a certain site.

  In the present specification, “hybridize” means that a certain nucleic acid molecule and another nucleic acid molecule specifically interact with each other under a certain condition to form a hybrid. Specifically, in the nucleic acid amplification reaction, it refers to a step in which the target nucleic acid or its equivalent and the nucleic acid equivalent corresponding to the amplification means form a hybrid and follow the subsequent amplification reaction. Hybridization is also referred to as “annealing” when specifically referred to herein. For hybridization in the amplification reaction, PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). See PCR Applications et al.

  In this specification, “annealing” means that a double-stranded nucleic acid that has been denatured into a single strand is returned to a double strand again. Therefore, in the nucleic acid amplification reaction, annealing and hybridization can be used in the same meaning. For example, after DNA is dissociated into single strands by heating or alkali treatment, and then slowly cooled or neutralized, it returns to a double strand state again. This phenomenon is called annealing, and this property can be used to examine the complementarity of base sequences between nucleic acid strands. When annealing is performed here, it can be determined that there is a degree of complementarity that can be used in the amplification method of the present invention.

  As used herein, a “hybridizable” nucleic acid or polynucleotide refers to a nucleic acid or polynucleotide that can hybridize to another nucleic acid or polynucleotide under the above hybridization conditions. Specifically, the hybridizable nucleic acid is a nucleic acid having at least 60% or more homology with a DNA base sequence encoding a polypeptide having the amino acid sequence shown in the sequence listing, preferably 80% or more of homology. And more preferably nucleic acids having a homology of 95% or more. Nucleic acid sequence homology is shown in score by using, for example, a search program BLAST using an algorithm developed by Altschul et al. (J. Mol. Biol. 215, 403-410 (1990)).

  As used herein, “highly stringent conditions” are designed to allow hybridization of DNA strands with a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA with significant mismatches. Say conditions. Hybridization stringency is primarily determined by temperature, ionic strength, and other additive conditions. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C. and the like. For such highly stringent conditions, see Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N, Y. 1989), 3rd edition (2001); and Anderson et al. Nucleic Acid Hybridization: A Practical Approach, IV, IRL Press Limited (Oxford, England). See Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, presence or absence of other additives, etc.) may be used. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples of such other agents are gelatin, Triton-X100, etc., but other suitable agents may also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization conditions typically include pH 6.8-7.4; however, under typical ionic strength conditions, the rate of hybridization is almost pH independent. PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). See PCR Applications et al. For general hybridization, see Anderson et al. , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).

Factors that affect nucleic acid duplex stability include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one of ordinary skill in the art to apply these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
Tm (° C) = 81.5 + 16.6 (log [Na ⁺ ]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na ⁺ ] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is the (guanine + in the hybrid Cytosine) base percentage. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch. In nucleic acid amplification, formamide is preferably not used in nucleic acid amplification. Accordingly, consideration of hybridization conditions in nucleic acid amplification can be used by those skilled in the art from the above general formula, modified according to the amplification reaction used.

  When RNA, PNA or the like is used, those skilled in the art can use a relational expression appropriate for the nucleotide. For RNA, see, for example, Freier S. et al. M.M. (1993) Hybridization: Considerations affecting Antisense Drugs in Antisense Research and Applications, eds. Grooke S. T.A. and Lebleu B. CRC Press, Inc. 67-82.

  It will be appreciated that those skilled in the art can find appropriate conditions for labeling or detection, etc. using the relational expressions as described above.

  Hybridizing nucleic acid molecules are often substantially complementary or completely complementary to each other.

  In this specification, “complementary” refers to a base sequence (nucleic acid) in which each base is replaced with a base paired by Watson-Crick bond in the base sequence of the nucleic acid when referring to the nucleic acid. . Usually, A and T (U) and C and G are said to be complementary to each other.

  As used herein, the term “complementary” or “substantially complementary” refers to a nucleic acid sequence that can specifically hybridize to another nucleic acid molecule. Complementary nucleic acid sequences are preferably 70% or more, 80% or more, 90% or more, 95%, 99% when one sequence is converted based on Watson-Crick base pairs, compared to the other sequence. It can have more or 100% sequence identity. Here, when this sequence identity is 100%, it is also said to be completely complementary.

  As used herein, the terms “nucleic acid”, “nucleic acid molecule”, “oligonucleotide”, “polynucleotide” are used interchangeably unless otherwise stated and are macromolecules composed of a series of nucleotides. (Polymer). In the present specification, an oligonucleotide refers to a single-stranded oligonucleotide unless otherwise specified.

  As used herein, “separation of nucleic acids” refers to separating a set of nucleic acids from a set of nucleic acids having different properties. Such separation can be carried out based on molecular weight, labeling, affinity and the like. When the molecular weight is separated by a clue, it can be separated by electrophoresis. Separation by molecular weight can also be performed using molecular sieves.

  In the present invention, the detection of the label can be performed directly or indirectly. When performing directly, for example, the radioactivity can be detected with a Geiger counter or the like, or the fluorescence can be directly detected with the naked eye or a camera. In the case of performing indirectly, for example, another label is bound to the label to detect the other label, or a factor that activates the label is added to activate the label (biotin, The detection can be performed by detecting the use of streptavidin or the like).

  As used herein, “autoradiograph” or “autoradiography” refers to the distribution of a substance in a living body by observing the uptake of a radioactive substance (tracer) in the living body using an X-ray photographic film or a dry plate. , A method of examining migration and metabolism by cytochemistry and histochemistry. Examples of autoradiographs include macro autoradiograph method (visible autoradiograph method), micro autoradiograph method (light microscope autoradiograph method), and ultramicro autoradiograph method (electron microscope autoradiograph method). In the present invention, any one can be selected and used according to the purpose.

  As long as the nucleic acid amplification method used in this specification can amplify a nucleic acid molecule, any method can be used, for example, polymerase chain reaction (PCR), ligase chain reaction (LCR), gap LCR. , Strand displacement amplification (SDA), Qβ replicase amplification, transcription amplification system (TAS), self-supported sequence replication system (3SR); nucleic acid sequence-based amplification (NASBA); transcription-mediated amplification (TMA), Chimeric Primer Initiated Nucleic Acid Amplification of Nucl (ICAN; Isothermally Primer Amplified of Nucl) ics acid) and loop-mediated isothermal amplification (LAMP) and the like.

  The polymerase chain reaction (PCR) is an amplification reaction in which amplification occurs by repeating three reactions: DNA double-strand dissociation, annealing with oligonucleotides, and complementary strand synthesis using primers and DNA polymerase. Say. Specific examples thereof are illustrated in the Examples herein. It is the most commonly used nucleic acid amplification technique. Saiki RK etal. (1985), Science, vol. 230, 1350-1354; Saiki RK etal. (1988), Science, vol. 239, 487-491; US 4,683,202, US 4,683,195, US 4, See 965,188.

  Ligase chain reaction (LCR) is a nucleic acid amplification reaction using ligase. When four adjacent 30 nt long probes complementary to the target DNA are subjected to a temperature cycle in which thermostable DNA ligase is present, Dependently, the ligation reaction between adjacent probes repeatedly occurs, and the ligation probes increase exponentially. The ligase chain reaction (LCR) method is a method for amplifying and detecting a target gene sequence by performing a temperature cycling reaction (repeated reaction of heat treatment and cooling) using a heat-resistant DNA ligase. In this method, the reaction is carried out using two types of adjacent gene fragments, that is, two types of adjacent gene fragments that are cooled and bound to both DNA double helices dissociated by heat treatment. Specifically, double-stranded DNA is heat-treated at 94-99 ° C., and each is dissociated into single strands. Subsequent cooling at 50-70 ° C. allows two adjacent gene fragments to bind to the target DNA separated into single strands. Furthermore, when the sequence is normal, adjacent gene fragments are linked by thermostable DNA ligase, but when even one base is abnormal, it is not linked. By repeating this series of reactions, the ligation product of gene fragments is amplified exponentially in the case of normal DNA sequences, but abnormal DNA sequences are not amplified. Landegren U. et al. (1988), Science, vol. 241, 1077-1080; Barany F. (1991), Proc. Natl. Acad. Sci. USA, vol. 88, 189-193; And JP-A-2001-269187.

  Gap LCR is one of nucleic acid amplifications using ligase, which introduces a gap of several bases at the ligation end, prevents false positives due to blunt end ligation between probes, and fills the gap between bound probes with DNA polymerase. Connect with See Marshall R.L. et al. (1994), PCR Methods Appl., Vol. 4, 80-84;

  Strand displacement amplification (SDA) is a nucleic acid amplification reaction using a DNA polymerase and a restriction enzyme lacking 5 ′ → 3 ′ exonuclease activity, and is extended by DNA polymerase by a single strand nick by restriction enzyme. A reaction in which the displaced DNA strand functions as a template for subsequent replication, resulting in a possible primer. It consists of two stages: target replication with Hinc II recognition site and SDA reaction amplification cycle. Since the SDA method amplifies nucleic acids at a single temperature, it is not necessary to expose the reaction system to a high temperature, and a plurality of targets can be amplified at the same time, so that automation is easy. The SDA method is a technology that amplifies DNA using the action of DNA polymerase that cuts the DNA of a pathogenic bacterium to be detected with a restriction enzyme and sequentially replaces the DNA fragments with the cut. It was developed as a method for amplifying genes by Becton Dickinson (BD). Walker G.T. et al. (1992), Proc. Natl. Acad. Sci. USA, vol.89, 392-396; Walker GT etal. (1992), Nucleic Acids Res., Vol.20, 1691-1696; See Japanese Patent Publication No. 7-114718.

  Qβ replicase amplification is a nucleic acid amplification reaction using an RNA-directed RNA polymerase (Qβ replicase), and an RNA probe containing a Qβ replicase recognition site is hybridized to a target and an unbound probe is used. This is a reaction in which the bound probe is replicated and amplified with Qβ replicase after removal. Miele EA etal. (1983), J. Mol. Biol., Vol. 171, 281-295; Lizardi PM et al. (1988), Biotechnology, vol. 6, 1197-1202; US 4,786,600, patent 2710159, patent See 3240151.

  Transcription amplification system (TAS) is a nucleic acid amplification reaction using reverse transcriptase and DNA-dependent RNA polymerase. Forward primer with the same sequence as target RNA, complementary to target RNA, and T7 RNA polymerase on the 5 'side. This reaction combines a step of obtaining a transcription product from a template RNA using a reverse primer with a promoter sequence, and a step of further synthesizing a transcription product using the obtained transcription product as a template. See Kwoh D.Y. etal. (1989), Proc. Natl. Acad. Sci. USA, vol.86 1173-1177;

  The self-supported sequence replication system (3SR) is a nucleic acid amplification reaction similar to TAS. However, by adding RNaseH to TAS, amplification of cDNA and RNA copy occurs and This is a template reaction. See Guatelli J.C. etal. (1990), Proc. Natl. Acad. Sci. USA, vol. 87, 1874-1878;

  Nucleic acid sequence-based amplification (NASBA) refers to a reaction using reverse transcriptase, RNase H, DNA-dependent RNA polymerase. Using a forward primer having the same sequence as the target RNA, a reverse primer complementary to the target RNA and having a 5'-side T7 RNA polymerase promoter sequence, and obtaining a transcript from the template RNA, and using the resulting transcript as a template The reaction further combines the steps of synthesizing the transcript. See Compton J. (1991), Nature, vol. 350, 91-92; Kievits T. (1991), J. Virol. Methods, vol. 35, 273-286; Patent 2648802, Patent 2650159.

  Transcription-mediated amplification (TMA) is a nucleic acid amplification reaction using reverse transcriptase and RNA polymerase having RNase H activity, and is a forward primer of the same sequence as the target RNA, complementary to the target RNA and T7 RNA on the 5 ′ side. This is a reaction combining a step of obtaining a transcription product from a template RNA using a reverse primer with a polymerase promoter sequence and a step of further synthesizing a transcription product using the obtained transcription product as a template. See Japanese Patent No. 3241717, Japanese Patent Laid-Open No. 11-46778, and Japanese Patent Laid-Open No. 2000-350592.

  Chimeric primer-initiated nucleic acid amplification (ICAN) under isothermal conditions is a nucleic acid amplification reaction using a DNA polymerase having a strand displacement activity, RNase H, and a strand displacement reaction using a chimeric primer composed of RNA-DNA. , Amplification reaction by template exchange reaction and nick introduction reaction. Notomi T. etal. (2000), Nucleic Acids Res., Vol. 28, e63; Nagamine K. etal. (2001), Clin. Chem., Vol. 47, 1742-1743; Japanese Patent No. 3313358, JP 2001-34790. See

  Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification reaction using a DNA polymerase having strand displacement activity. The 3 ′ end of the synthesized DNA always forms a loop, and the next DNA This is a reaction to design a primer to be a starting point for synthesis. http://www.takara.co.jp/news/2000/07-09/00-i-019.htm; see WO2000-56877.

  As used herein, “nucleic acid amplification conditions” refers to any conditions under which nucleic acid amplification occurs in a nucleic acid amplification method to be used. Such conditions can be easily selected by those skilled in the art with reference to the above-mentioned literature.

  In the present specification, a nucleic acid molecule used as a primer pair is composed of a “forward single stranded nucleic acid molecule” and a “reverse single stranded nucleic acid molecule”. Usually, in order to amplify a certain nucleic acid molecule, the forward direction refers to the direction from the 5 'end to the 3' end of the single strand or sense strand, and the reverse direction refers to the opposite.

  In the present specification, the “primer” refers to a substance necessary for initiation of a reaction of a polymer compound to be synthesized in a polymer synthase reaction. In the synthesis reaction of a nucleic acid molecule, a nucleic acid molecule (for example, DNA or RNA) complementary to a partial sequence of a polymer compound to be synthesized can be used. The length of the primer is usually 100 bases or less, more preferably 8 bases to 80 bases, and even more preferably 10 bases to 40 bases, but is not limited thereto.

  As a nucleic acid molecule usually used as a primer in the field of genetic engineering, it hybridizes with a nucleic acid sequence of a target gene under specific hybridization conditions (usually nucleic acid amplification conditions), preferably substantially, Those having a complementary nucleic acid sequence of at least 8 consecutive nucleotides in length. Such a nucleic acid sequence is preferably at least 9 contiguous nucleotides long, more preferably 10 contiguous nucleotides long, even more preferably 11 contiguous nucleotides long, 12 contiguous nucleotides long, 13 19 contiguous nucleotide lengths, 14 contiguous nucleotide lengths, 15 contiguous nucleotide lengths, 16 contiguous nucleotide lengths, 17 contiguous nucleotide lengths, 18 contiguous nucleotide lengths, 19 contiguous lengths It can be a nucleic acid sequence of nucleotide length, 20 contiguous nucleotide lengths, 25 contiguous nucleotide lengths, 30 contiguous nucleotide lengths, 40 contiguous nucleotide lengths, 50 contiguous nucleotide lengths. Nucleic acid sequences used as probes include nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, 95% homologous to the sequences described above. It is. A sequence suitable as a primer may vary depending on the nature of the sequence intended for synthesis (amplification), but those skilled in the art can appropriately design a primer according to the intended sequence. Such primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar). An amplification product used in the present invention can be prepared using such a primer.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such a number may be a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, it can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

  As used herein, “template” refers to a nucleic acid molecule containing a sequence to be amplified in a nucleic acid amplification reaction or an equivalent thereof.

  In this specification, when referring to genetic manipulation, “vector” or “recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence to a target cell. Such vectors can be autonomously replicated in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals and individual plants, or can be integrated into chromosomes. Examples include those containing a promoter at a position suitable for nucleotide transcription.

  In the present specification, among vectors, a vector suitable for cloning is referred to as a “cloning vector”. Such cloning vectors usually contain multiple cloning sites that contain multiple restriction enzyme sites. Such restriction enzyme sites and multiple cloning sites are well known in the art, and those skilled in the art can appropriately select and use them according to the purpose. Such techniques are described in the literature described herein (eg, Sambrook et al., Below).

  As used herein, “expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. Regulatory elements can preferably include terminators, selectable markers such as drug resistance genes, and enhancers. It is well known to those skilled in the art that the type of expression vector of an organism (eg, animal) and the type of regulatory element used can vary depending on the host cell.

Examples of recombinant vectors for prokaryotic cells include pcDNA3 (+), pBluescript-SK (+/-), pGEM-T, pEF-BOS, pEGFP, pHAT, pUC18, pFT-DEST ^™ 42GATEWAY (Invitrogen) .

  Recombinant vectors for animal cells include pcDNAI / Amp, pcDNAI, pCDM8 (all available from Funakoshi), pAGE107 [JP-A-3-229 (Invitrogen), pAGE103 [J. Biochem. , 101, 1307 (1987)], pAMo, pAMoA [J. Biol. Chem. , 268, 22782-2787 (1993)], retroviral expression vectors based on Murine Stem Cell Virus (MSCV), pEF-BOS, pEGFP and the like.

  Recombinant vectors for plant cells include, but are not limited to, pPCVICEn4HPT, pCGN1548, pCGN1549, pBI221, pBI121 and the like.

  As used herein, “operably linked” means under the control of a transcriptional translational regulatory sequence (eg, promoter, enhancer, silencer, etc.) or translational regulatory sequence that has the expression (operation) of a desired sequence. To be placed. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique for introducing a nucleic acid molecule into a cell may be used, and examples thereof include transformation, transduction, transfection, and the like. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J, et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  Moreover, as a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used. For example, transfection, transduction, transformation, etc. (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol. , 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

In the present specification, the “gene introduction reagent” refers to a reagent used for promoting introduction efficiency in a method for introducing a nucleic acid (normally encoding a gene, but not limited thereto). Examples of such gene introduction reagents include, but are not limited to, cationic polymers, cationic lipids, polyamine reagents, polyimine reagents, calcium phosphate, and the like. Specific examples of reagents used in transfection include reagents commercially available from various sources, such as Effectene Transfection Reagent (cat.no.301425, Qiagen, CA), TransFast ™ Transfection Reagent (E2431). , Promega, WI), Tfx ^™ -20 Reagent (E2391, Promega, WI), SuperFect Transfection Reagent (301305, Qiagen, CA), PolyFect Transfection Reagent (301105, Qenten, CA9, Regent A 116, Q9, 2000, Q9, 2000, Q9, 2000 en corporation, CA), JetPEI (× 4) conc. (101-30, Polyplus-translation, France) and ExGen 500 (R0511, Fermentas Inc., MD) and the like. In the present invention, such a gene introduction reagent can be used when introducing the nucleic acid molecule of the present invention into a cell.

  As used herein, “transformant” refers to all or part of a living organism such as a cell produced by transformation (such as a tissue). Examples of the transformant include all or a part (tissue or the like) of living organisms such as cells of prokaryotes, yeasts, animals, plants, insects and the like. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject. The cell used in the present invention may be a transformant.

  When a prokaryotic cell is used in the present invention for genetic manipulation or the like, the prokaryotic cell may be a prokaryotic cell belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc. Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1 are exemplified. Or in this invention, the cell isolate | separated from the natural product can also be used.

  Examples of animal cells that can be used in genetic manipulation and the like in the present specification include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells that are Chinese hamster cells, BHK cells, African green monkey kidney cells, humans Examples include leukemia cells, HBT5637 (Japanese Patent Laid-Open No. 63-299), and human colon cancer cell lines. Mouse myeloma cells such as ps20, NSO, rat myeloma cells such as YB2 / 0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), human leukemia cells such as BALL-1, Africa COS-1, COS-7 as the kidney monkey cell, HCT-15 as the human colon cancer cell line, human neuroblastoma SK-N-SH, SK-N-SH-5Y, mouse neuroblastoma Neuro2A, etc. Is exemplified. Alternatively, primary cultured cells can also be used in the present invention.

  Plant cells that can be used in the present specification for genetic manipulation and the like include callus or a part thereof and suspension culture cells, solanaceae, gramineous, cruciferous, rose, legume, cucurbitaceae, perilla, lily , But not limited to, cells of plants such as red crustaceae and ceraceae.

  As used herein, “detection” or “quantification” of gene expression (eg, mRNA expression, polypeptide expression) can be accomplished using appropriate methods, including, for example, mRNA measurement and immunoassay methods. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR. Examples of the immunological measurement method include ELISA method using a microtiter plate, RIA method, fluorescent antibody method, Western blot method, immunohistochemical staining method and the like. Examples of the quantitative method include an ELISA method and an RIA method. It can also be performed by a gene analysis method using an array (eg, DNA array, protein array). The DNA array is widely outlined in (edited by Shujunsha, separate volume of cell engineering "DNA microarray and latest PCR method"). For protein arrays, see Nat Genet. 2002 Dec; 32 Suppl: 526-32. Examples of gene expression analysis methods include, but are not limited to, RT-PCR, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, in vitro translation and the like. Such further analysis methods are described in, for example, Genome Analysis Experimental Method / Yusuke Nakamura Lab Manual, Editing / Yusuke Nakamura Yodosha (2002), etc., all of which are incorporated herein by reference. Is done.

  As used herein, the term “kit” refers to a unit in which parts to be provided (eg, reagents, particles, etc.) are usually divided into two or more compartments. This kit form is preferred when it is intended to provide a composition that should not be provided in a mixed form but is preferably used in a mixed state immediately prior to use. Such kits preferably include instructions that describe how to treat the provided parts (eg, reagents, particles, etc.). Such a manual may be any medium. Examples of such a medium include, but are not limited to, a paper medium, a transmission medium, and a recording medium. Examples of the transmission medium include, but are not limited to, the Internet, an intranet, an extranet, and a LAN. Examples of the recording medium include, but are not limited to, CD-ROM, CD-R, flexible disk, DVD-ROM, MD, mini disk, MO, and memory stick.

(screening)
As used herein, “screening” refers to selecting a target such as a target organism or substance having a specific property from a large number of populations by a specific operation / evaluation method. For screening, the method or nucleic acid construct of the present invention can be used. In the present invention, since any nucleic acid construct can be freely produced, the present invention exhibits a remarkable effect also in screening.

(Gene therapy)
The use of the present invention includes incorporating a gene for gene therapy to cells by modifying a freely prepared nucleic acid molecule. The gene can be placed under the control of a tissue-specific promoter, or under the control of a constitutive promoter, or one or more other expression control regions for expression of the gene in cells that require the gene. Examples of genes used for gene therapy include CFTR gene for cystic fibrosis, α-1-antitrypsin for lung disease, adenosine deaminase (ADA) for immune system disease, blood cell disease Such as, but not limited to, Factor IX and Interleukin-2 (IL-2), and Tumor Necrosis Factor (TNF) for cancer treatment.

  Gene sequences that can be used for gene therapy can be obtained by searching in known databases such as GenBank, DDBJ, and EMBL.

  Furthermore, the present invention may be used as part of a process for working with or screening a library, for assessing sequence function, or for screening protein expression, or for specific proteins or for specific cell types. It can be used to evaluate the effect on a specific expression control region.

  As used herein, “at least one tip” of a nucleic acid means at least one of the 3 ′ end or the 5 ′ end of the nucleic acid.

  In the present specification, the “complex” refers to a state in which two or more molecules associate with each other through an interaction and behave as if they were one molecule. Thus, the hybridized nucleic acid-nucleic acid is a complex.

  As used herein, “nucleic acid synthase” refers to any enzyme having the ability to synthesize nucleic acids. In this specification, it is also called a polymerase. A generic term for enzymes that catalyze the reaction of condensing nucleotides into polynucleotides. Examples of such a nucleic acid synthase include, but are not limited to, DNA polymerase (eg, DNA-dependent DNA polymerase, RNA-dependent DNA polymerase), RNA polymerase, and the like.

  Examples of the polymerase include DNA-dependent DNA polymerase and RNA-dependent DNA polymerase. In addition, it is understood that DNA or RNA can be detected by either polymerase. The only difference is which mold is either (or both). (RNA-dependent DNA polymerases also use DNA as a template. Some of the DNA-dependent DNA polymerases sold also have RNA-dependent activity). Examples of such polymerase include Escherichia coli-derived DNA polymerase I, DNA polymerase I Klenow fragment, Taq polymerase, KLA-Taq polymerase, KOD polymerase, Vent polymerase, AMV reverse transcriptase, Pfu polymerase, T4 DNA polymerase, and the like. But are not limited to these.

  As used herein, “extension reaction” refers to any reaction in which a nucleic acid extends at least one nucleotide. When an extension reaction using a polymerase is performed, since a nucleotide is usually introduced based on a template, a specific sequence is extended in a nucleic acid to be extended such as a nucleic acid to be labeled.

  The extension reaction consists of (i) a hybridization (annealing) reaction between the target nucleic acid and its complementary strand and a nucleic acid primer and a nucleic acid probe, and (ii) a primer strand extension reaction and a probe decomposition reaction by a nucleic acid synthase. (I) and (ii) can be carried out in an aqueous solution containing a suitable buffer such as a Tris-HCl buffer. This hybridization reaction can be typically performed at 55 to 75 ° C., but is not limited thereto. The enzyme reaction can be typically performed at 37 ° C., but is not limited thereto.

  The denaturation of the primer extension product can be carried out, for example, by subjecting the solution containing the primer chain extension product obtained by the extension reaction to a heat treatment at 94 to 95 ° C. for 0.5 to 1 minute, for example.

For amplification of the target nucleic acid, for example, the above-described extension reaction and denaturation may be repeated a plurality of times under the same conditions as described above until the target nucleic acid (labeled nucleic acid) reaches a desired amount. If the above process is performed n times, the target nucleic acid amount is theoretically amplified to 2 ⁿ -1 times the original.

  Such an extension reaction can be carried out simply and efficiently by using a commercially available apparatus for PCR (polymerase chain reaction) (for example, sold by Applied Biosystems).

  Nucleic acid can be removed from the mixture using any method in the art. Examples of such extraction include, but are not limited to, electrophoresis, chromatography, denaturation, alcohol precipitation, affinity purification, and antibodies.

  The step of providing a nucleic acid used in the method of the present invention can be performed using any technique known in the art (for example, chemical synthesis, utilization of genetic engineering, extraction from a living body).

  The production of the complex used in the method of the present invention can be performed using any technique well known in the art. For example, it is understood that complex formation can be performed by appropriately selecting hybridization conditions that are normally used as described elsewhere in this specification.

  The extension reaction used in the method of the present invention can be performed using any technique known in the art. The extension reaction includes an extension reaction catalyst (for example, a nucleic acid synthase), a substance that provides conditions for the reaction of the catalyst (for example, a buffer solution), and a nucleotide to be entered during the extension (labeled, unlabeled, etc.) ) Can be considered. Moreover, in order to control reaction conditions, you may use the apparatus (for example, thermostat) which adjusts temperature etc.

  After the extension reaction, the step of removing the nucleic acid with the label introduced from the mixture can also be performed using any technique known in the art. Such a removal step can be performed by any technique that is commonly used for purifying or separating nucleic acids.

  In the method of the present invention, in the step of detecting the presence of an extension product corresponding to the target nucleic acid, the detection is carried out by any method directly or indirectly using any technique well known in the art. It is understood that you can. Here, it is understood that the presence of the extension product is an indicator of the presence of the target nucleic acid. This is because the extension product is not detected unless there is an unknown nucleic acid before extension. However, since it is considered that there are a plurality of types of unknown nucleic acids before being extended, more detailed detection can be performed by determining the sequence of the extension product. Such extension products can be sequenced using any technique known in the art.

  It is preferable that the complementarity between the nucleic acid molecules to be hybridized is sufficient to allow the extension reaction to proceed. The degree of complementarity varies depending on the length, extension reaction conditions, and the like, and the vicinity of the 3 'end is preferably suitable for the extension reaction. In particular, it may be preferred that 1-2 nucleotides at the 3 'end have perfect complementarity. Alternatively, it is usually preferable that the nucleic acid has at least 50% homology based on the shorter nucleic acid, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably It may further be preferred that have a homology of at least 90%, more preferably at least 95%. Alternatively, it may be complementary to all the nucleic acids to be labeled.

  The DNA polymerase that can be used in the method of the present invention includes all DNA polymerases having an endogenous exonuclease activity, preferably 3′-5 ′ exonuclease activity, which can perform the DNA ligation reaction of the present invention. . Such polymerases can be identified by assaying for the ability to link two linear DNA molecules having ends with complementary nucleotide sequences, as described herein. Preferably, the DNA polymerase is selected from the group consisting of vaccinia virus DNA polymerase, T4 DNA polymerase, and Klenow fragment of E. coli DNA polymerase I. In selecting a DNA polymerase, it is particularly important to consider the accuracy of the synthesis (extension) reaction. Therefore, particularly in the tag region, an α-type enzyme (eg, KOD-plus-: TOYOBO) having strong 3 ′ → 5 ′ exonuclease activity and high-fidelity is desirable. Preferably, a highly accurate enzyme such as KOD-plus-: TOYOBO is used as the DNA polymerase. The length of the complementary nucleotide sequence of each linear DNA molecule can be between about 5 and about 100 nucleotides, preferably between about 8 and about 50 nucleotides, and most preferably between about 10 and 35 nucleotides. Various known stimulating factors may be added to the incubation mixture. As such a stimulating factor, a factor that improves the efficiency of the reaction and stabilizes a desired reaction product, for example, a single-stranded DNA binding protein, can be used.

  Various known stimulators may be added to the incubation mixture to increase the efficiency of the reaction and / or to stabilize the reaction product. Suitable stimulators include single stranded DNA binding proteins. Among the single-stranded DNA binding proteins that can be used are vaccinia and E. coli single-stranded binding proteins, herpes simplex virus ICP8 protein, and yeast and human replicating protein A (eg, yRPA and hRPA). Some include but are not limited to these. One skilled in the art will readily be able to identify other suitable stimulators that can be usefully used in combination with DNA polymerases in the methods of the invention. In one embodiment of the invention, vaccinia single-stranded binding protein is used to increase the yield of DNA ligation reaction using vaccinia DNA polymerase.

  The degree of complementarity between the ends of a linear DNA molecule can vary between about 5 and about 100 nucleotides, preferably between about 8 and about 50 nucleotides, and most preferably between about 10 and 35 nucleotides. Linear DNA molecules that can be ligated using the methods of the present invention can be obtained using restriction enzyme cleavage from various vectors including genomic DNA of prokaryotic or eukaryotic genomes, as well as plasmids, cosmids, phages, BACs, and the like. You may obtain it. Alternatively, the linear DNA molecule may be synthesized by a chemical method using, for example, automatic synthesis. DNA molecules may be derived from RNA species such as mRNA, tRNA and rRNA from any species, and first converted to cDNA by reverse transcriptase and amplified as described in Sambrook et al. (1989) (below). can do.

  Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associate ES and Wiley-Interscience; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer & Expression Analysis”, Yodosha, 1997, etc., which are related in this specification (may be all) Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. etal. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

  In order to confirm the presence of the nucleic acid in the present specification, it can be evaluated by any appropriate method including a molecular biological measurement method such as radioactivity method, fluorescence method, Northern blot method, dot blot method, PCR method and the like.

  Various methods for producing transgenic organisms are described, for example, in M.M. Markkula et al., Rev. Reprod. 1, 97-106 (1996); T.A. Wall et al. Dairy Sci. 80, 2213-2224 (1997); C. Dalton, et al., Adv. Exp. Med. Biol. , 411, 419-428 (1997); Lubon et al., Transfus. Med. Rev. , 10, 131-143 (1996), but not limited thereto. Each of these documents is incorporated herein by reference.

(kit)
As used herein, the term “kit” refers to a unit provided with a portion (eg, reagent, enzyme, template nucleic acid, standard, etc.) to be provided, usually divided into two or more compartments. This kit form is preferred when it is intended to provide a composition that should not be provided in a mixed form but is preferably used in a mixed state immediately prior to use. Such kits preferably do what is provided with the moieties (eg, reagents, enzymes, nucleotides, labeled nucleotides, nucleotides that stop the extension reaction, etc. (and their triphosphates), template nucleic acids, standards, etc.) It is advantageous to have instructions describing what should be processed. In the present specification, when a kit is used as a reagent kit, the kit usually includes reagent components, a buffer solution, a salt concentrate, auxiliary means for use, instructions describing the method of use, and the like.

  In the present specification, the “instruction” describes the usage method, reaction method and the like of the reagent of the present invention to the user. This instruction manual describes a word indicating a procedure such as an enzyme reaction of the present invention. This instruction is prepared according to the format prescribed by the national supervisory authority where the present invention is implemented as necessary, and it is clearly stated that it has been approved by the supervisory authority. The instruction sheet is a so-called package insert, and is usually provided as a paper medium, but is not limited thereto. For example, a film attached to a bottle, an electronic medium (for example, a home page provided on the Internet) (Website), e-mail).

(program)
The program provided by the present invention will be described below. The description of the program can include any language in the field (for example, C + language, Perl, Basic, html, XML, Pascal, FORTRAN, etc.). It is not limited to. It should be understood that the term “program” as used herein refers to a computer program unless otherwise specified.

(recoding media)
The recording medium provided by the present invention will be described below. As the recording medium, any form (for example, flexible disk, MO, CD-ROM, CD-R, DVD-ROM) can be used as long as it can record a program. It is understood that any type (such as a memory card) can be used.

(system)
An example of a computer configuration for executing the design method of the present invention or a system for realizing the same will be described with reference to FIG. FIG. 4 shows a configuration example of a computer 500 for executing the method of the present invention.

  The computer 500 includes an input unit 501, a CPU 502, an output unit 503, a memory 504, and a bus 505. The input unit 501, the CPU 502, the output unit 503, and the memory 504 are connected to each other via a bus 505. The input unit 501 and the output unit 503 are connected to the input / output device 506.

  An outline of the processing of the present invention executed by the computer 500 will be described.

  A program for executing the method of the present invention is stored in the memory 502, for example. Alternatively, the program for executing the method of the present invention can be recorded on any type of recording medium such as a flexible disk, MO, CD-ROM, CD-R, DVD-ROM, independently or together. Alternatively, it may be stored in the application server. The program of the present invention recorded on such a recording medium is loaded into the memory 504 of the computer 500 via the input / output device 506 (for example, a disk drive, network (for example, the Internet)). When the CPU 502 executes the program of the present invention, the computer 500 functions as a device that executes the processing of the method of the present invention.

  Information related to the system used in the present invention is input via the input unit 501. Also, the measured descriptor data is input. Information related to known information may be input as necessary.

  The CPU 502 generates display data from the information generated in the present invention based on the information input by the input unit 501, and stores the display data in the memory 504. Thereafter, the CPU 502 can store these pieces of information in the memory 504. Thereafter, the output unit 503 outputs the array selected by the CPU 502 as display data. The output data can be output from the input / output device 506.

Detailed Description of Preferred Embodiments of the Invention
The description of the preferred embodiment is described below, but it should be understood that this embodiment is an illustration of the present invention and that the scope of the present invention is not limited to such a preferred embodiment.

  In one aspect, the present invention is a method for producing a nucleic acid construct in which a plurality of nucleic acid molecules are linked, the method comprising the following: (a) a first nucleic acid molecule is amplified to a first double strand Obtaining an amplification product; (b) amplifying a second nucleic acid molecule to obtain a second double-stranded amplification product, wherein the 3 ′ end portion of the first double-stranded amplification product; (C) step (a), wherein the 5 ′ end portion of the second double-stranded amplification product is amplified so as to have a sequence that hybridizes under conditions where only a specific sequence hybridizes; And mixing the first and second double-stranded amplification products obtained by (b), denaturing them into single strands, and hybridizing under conditions where only the specific sequences hybridize; and (D) Nucleic acid amplification using the mixture of (c) above as a template Comprising the step of obtaining a connecting nucleic acid amplification product undergoes a reaction to provide a method. Here, two parallel amplification reactions are carried out. The 3 ′ end portion of the first double-stranded amplification product and the 5 ′ end portion of the second double-stranded amplification product contain only specific sequences. It is characterized by being amplified to have a sequence that hybridizes under hybridizing conditions. Such amplification can be achieved by designing the primer to have a specific sequence.

  In one embodiment, the method of the invention comprises: (a) using a first nucleic acid molecule as a template and a first reverse one capable of hybridizing under nucleic acid amplification conditions to the 3 ′ end portion of the first nucleic acid molecule. A nucleic acid amplification reaction is carried out using a single-stranded nucleic acid molecule and a first forward single-stranded nucleic acid molecule that can be hybridized under the above-mentioned nucleic acid amplification conditions as a primer to the 5 ′ terminal portion of the complementary strand of the first nucleic acid molecule A step of obtaining a first double-stranded amplification product; (b) using a second nucleic acid molecule as a template, a second nucleic acid molecule capable of hybridizing to the 3 ′ end portion of the second nucleic acid molecule under the nucleic acid amplification conditions described above; Using as a primer a reverse single-stranded nucleic acid molecule and a second forward single-stranded nucleic acid molecule that can hybridize under the nucleic acid amplification conditions to the 5 ′ end portion of the complementary strand of the second nucleic acid molecule Performing a nucleic acid amplification reaction to obtain a second double-stranded amplification product, wherein the first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are The 3 ′ end portion of the first double-stranded amplification product and the 5 ′ end portion of the second double-stranded amplification product are designed to hybridize under conditions where only specific sequences hybridize. (C) The first and second double-stranded amplification products obtained in steps (a) and (b) are mixed, denatured into single strands, and only specific sequences are hybridized. And (d) using the mixture of (c) as a template, the first reverse single-stranded nucleic acid molecule, and the second forward single-stranded nucleic acid molecule. Perform nucleic acid amplification reaction using as a primer, Comprising the step of obtaining a.

  In one preferred embodiment, the present invention further comprises the step of (e) amplifying the double stranded nucleic acid molecule obtained by step (d).

  In one embodiment, the reverse single stranded nucleic acid molecule and the forward single stranded nucleic acid molecule have sequences and lengths sufficient to specifically hybridize and do not interfere with the amplification reaction. Such a length can be, for example, at least 15 nucleotides in length, for example, 40 to 80 nucleotides in length.

  In one embodiment, the first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule used in the present invention are the first nucleic acid molecule and the second nucleic acid molecule, A tag sequence that does not hybridize under the condition that only the specific sequence hybridizes and a tag complementary sequence that hybridizes under the condition that only the specific sequence hybridizes to the tag sequence may be included.

  A representative example of this embodiment will be described with reference to FIGS.

  Here, the first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are hybridized only with a specific sequence in both the first nucleic acid molecule and the second nucleic acid molecule. And a tag complementary sequence complementary to the tag sequence.

  In this embodiment, the double-stranded nucleic acid molecule A has a forward primer PA (→) that can specifically hybridize to a sequence complementary to the nucleic acid molecule A and a reverse direction that can specifically hybridize to the nucleic acid molecule A. Amplified by PCR reaction using primer PA (←). The double-stranded nucleic acid molecule B includes a forward primer PB (→) that can specifically hybridize to a sequence complementary to the nucleic acid molecule B and a reverse primer PB (specifically hybridizable to the nucleic acid molecule B). It is amplified by the PCR reaction using ←) (FIG. 2 (a)).

  Here, the primer PA (←) and the primer PB (→) each have a tag sequence portion (TA (←) and TB (→)), and TA (←) and TB (→) are substantially Complementary.

  The amplification products in steps (a) and (b) are mixed, heat-denatured and quenched to anneal complementary nucleic acids. Here, as shown in FIG. 3, four types of hybrids are formed. Here, DNA polymerase and nucleotide bases are added to synthesize the complementary strand of the single-stranded nucleic acid moiety in the formed hybrid III and hybrid IV (FIG. 3), and nucleic acid molecule A, complementary tag moiety, and nucleic acid molecule B are A ligated nucleic acid construct is obtained.

  Thereafter, if necessary, further PCR reaction is performed using the primer PA (→) and the primer PB (→) to amplify the target nucleic acid construct.

  Here, since the complementary tag portion is incorporated into the final product, it is important to use a sequence that does not interfere with the purpose. For example, in one embodiment, the tag sequence portion is the sequence of the terminal portion of the nucleic acid molecule A or nucleic acid molecule B on the linking portion side.

  In addition, when the ends of the amplification product are not smooth, it may cause the step (d) to be hindered or may cause an undesired mutation in the target nucleic acid construct. Therefore, it is desirable that the ends of the amplification products are smooth (that is, the double strands have exactly the same length and the ends are aligned). For example, since Taq polymerase adds A to the 3 'end, it is not desirable as an enzyme in the PCR reaction of step (a). Examples of a desirable enzyme for the PCR in step (a) include KOD-Plus- (available from TOYOBO).

  Further, in the step (c), it is empirically important to sufficiently mix in the annealing step after the modification.

  In another embodiment, the first forward single stranded nucleic acid molecule has a sequence that hybridizes with the 5 ′ end portion of the second nucleic acid molecule under conditions where only specific sequences hybridize, or The two reverse single-stranded nucleic acid molecules may have a sequence that hybridizes with the 3 ′ end portion of the first nucleic acid molecule under conditions where only a specific sequence hybridizes, or both. . In this case, a tag sequence is not required, but a tag sequence may be used. In order not to introduce a foreign sequence between a plurality of sequences to be linked, it is preferable to use a form that does not include this tag sequence. However, even if there is a complementary sequence between the template nucleic acid molecule and the primer as in this embodiment, a specific design (for example, introduction of restriction sites, introduction of introns, For example, introduction of an intein site may be performed.

  In this embodiment, preferably, the first forward single-stranded nucleic acid molecule has a sequence complementary to the 5 ′ end portion of the second nucleic acid molecule, or the second reverse single-stranded nucleic acid. The molecule may have a sequence complementary to the 3 ′ end portion of the first nucleic acid molecule, or both.

  In alternative embodiments, the amplification step in the method according to the invention may use other amplification methods known to those skilled in the art. Alternative amplification methods include ligase chain reaction (LCR) method (Barany, Proc. Natl. Acad. Sci. USA 88 189 (1991)), gap LCR, strand displacement amplification (SDA) method (Walker et al., Nucl. Acid.Res.20: 1691 (1992)), Qβ replicase amplification, transcription amplification system (TAS), self-supported sequence replication system (3SR), nucleic acids using chimeric oligonucleotide primers under isothermal conditions Sequence amplification (ICAN) method (Japan Patent No. 3433929) Loop-mediated isothermal amplification (LAMP) (Loop-mediated thermal amplification) method (Tsuguniori Notomi et al. (2000) Nucleic Acids Research, Vol. 28, No. 12: e63), and the like. However, in the method according to the present invention, the most preferred amplification method is the PCR method.

  In an alternative embodiment, the nucleic acid molecule A has an overlapping sequence with the nucleic acid molecule B at the terminal portion on the side linked to the nucleic acid molecule B, and can be linked by this portion in step (c). In this case, the primer PA (←) and the primer PB (→) do not need to have tag portions complementary to each other. Such an embodiment can be used, for example, to obtain a clone containing a full-length cDNA from clones of the same cDNA isolated separately by screening.

  In an alternative embodiment according to the present invention, the linked nucleic acid molecules can be 3 or more.

  In one embodiment, the present invention may provide a kit for making a nucleic acid construct according to the present invention. The kit according to the present invention comprises a single-stranded nucleic acid molecule that can be used as a forward and / or reverse primer in steps (a) and (b), optionally including other components. Also good. These components include, but are not limited to, reaction buffers, enzymes, oligonucleotides, sterile water, instructions for use, and the like. These components can be provided in the same container or in separate containers as required.

  In another aspect, the present invention provides a nucleic acid construct obtained by the nucleic acid amplification method of the present invention. The present invention also provides a polypeptide encoded by the nucleic acid construct provided by the present invention.

  In another aspect, the present invention provides two nucleic acid primer sets used for carrying out the nucleic acid ligation method of the present invention. The set includes a first forward single stranded nucleic acid molecule and a first reverse single stranded nucleic acid molecule for amplifying the first nucleic acid molecule to obtain a first double stranded amplification product. A first primer set, a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule for amplifying the second nucleic acid molecule to obtain a second double-stranded amplification product; A first forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule comprising: a 3 ′ end portion of the first double-stranded amplification product; The second double-stranded amplification product is designed to hybridize with the 5 ′ end portion under conditions where only the specific sequence hybridizes. Specific conditions are as described above in this specification and illustrated in the examples.

  In another aspect, the present invention provides a method for designing two nucleic acid primer sets, comprising A) comparing a nucleic acid sequence of a first nucleic acid molecule and a second nucleic acid molecule, Determining whether each of the nucleic acid molecule and the second nucleic acid molecule have internal hybridizing sequences that hybridize under conditions in which only specific sequences hybridize; B) to amplify the first nucleic acid molecule A first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule, and a second forward one for amplifying the second nucleic acid molecule. A step of designing a second primer set comprising a single-stranded nucleic acid molecule and a second reverse-direction single-stranded nucleic acid molecule, wherein in the step A), when it is determined that the hybridizing sequence does not exist, Hybridize A first tag nucleic acid sequence and a second tag nucleic acid sequence that hybridize to each other under the condition, respectively, upstream of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule at the 5 ′ end, respectively. In the step A), when it is determined that the hybridizing sequence is present, only the specific sequence hybridizes to the internal hybridizing sequence and the internal hybridizing sequence. An internal hybridizing complementary sequence that hybridizes under conditions, or the first tag nucleic acid sequence and the second tag nucleic acid sequence, respectively, and the first reverse single-stranded nucleic acid molecule and the second forward one Designing a primer to include upstream of the 5 ′ end of the single stranded nucleic acid molecule. Here, the length of the sequence to be designed and the sequence itself can be selected using conventional knowledge in the art in primer design. Such designs are described, for example, in Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995).

  In another aspect, the present invention provides a method for producing two nucleic acid primer sets, comprising: A) comparing the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule, Determining whether each of the nucleic acid molecule and the second nucleic acid molecule have internal hybridizing sequences that hybridize under conditions in which only specific sequences hybridize; B) to amplify the first nucleic acid molecule A first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule, and a second forward one for amplifying the second nucleic acid molecule. A step of designing a second primer set comprising a single-stranded nucleic acid molecule and a second reverse-direction single-stranded nucleic acid molecule, wherein in the step A), when it is determined that the hybridizing sequence does not exist, Hybridize A first tag nucleic acid sequence and a second tag nucleic acid sequence that hybridize to each other under the conditions of the first reverse single-stranded nucleic acid molecule and the 5 ′ end of the second forward single-stranded nucleic acid molecule, respectively. Primers are designed to be included upstream, and in step A), when it is determined that the hybridizing sequence is present, only the specific sequence hybridizes to the internal hybridizing sequence and the internal hybridizing sequence. An internal hybridizing complementary sequence that hybridizes under soy conditions, or the first tag nucleic acid sequence and the second tag nucleic acid sequence, respectively, the first reverse single-stranded nucleic acid molecule and the second forward direction, respectively. Designing a primer to include upstream of the 5 ′ end of the single-stranded nucleic acid molecule; and C) having the sequence of the designed primer. Comprising the step of producing a nucleic acid molecule that provides methods. Here, the steps A) and B) can take any form described above. Next, it will be understood by those skilled in the art that the step C) can utilize any nucleic acid molecule amplification / production method known in the art.

  In another embodiment, the present invention can also include computer software for designing single stranded nucleic acid molecules that can be used as forward and / or reverse primers according to the present invention. These computer softwares can include any software that designs primers suitable for the present invention. A synthesizer for synthesizing the designed primer is also included in the scope of the present invention.

  In one aspect, the present invention is a program for designing two nucleic acid primer sets, and A) compares a nucleic acid sequence of a first nucleic acid molecule and a second nucleic acid molecule with a nucleic acid sequence determination means A procedure for determining whether the first nucleic acid molecule and the second nucleic acid molecule have internal hybridizing sequences that hybridize under conditions in which only specific sequences hybridize to each other; A first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule; and the second nucleic acid molecule A procedure for designing a second primer set including a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule for amplification, wherein the hybridization is performed in the step A). Arrangement Is determined to be present, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize to each other under the hybridizing conditions are converted into the first reverse single-stranded nucleic acid molecule and the second tag nucleic acid molecule, respectively. The primer is designed so as to be included upstream of the 5 ′ end of the forward single-stranded nucleic acid molecule of the above, and when it is determined in the above step A) that the hybridizing sequence is present, the internal hybridizing sequence and the internal hybridizing sequence are determined. An internal hybridizing complementary sequence that hybridizes under conditions where only the specific sequence hybridizes to a soybean sequence, or the first tag nucleic acid sequence and the second tag nucleic acid sequence, respectively, in the first reverse direction. Primers are designed to be included upstream of the 5 ′ end of the single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule. Procedure, including, provides a program. Here, as described in the above method, each procedure can be performed by designing primers using any known method.

  Accordingly, in another aspect, the present invention is a recording medium storing a program for designing two nucleic acid primer sets, wherein the program includes A) a first nucleic acid molecule in the nucleic acid sequence determination means. And whether the first nucleic acid molecule and the second nucleic acid molecule have internal hybridizing sequences that hybridize under conditions where only specific sequences hybridize to each other. A step of determining whether or not: B) the primer design means includes a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule; A procedure for designing a second primer set including one primer set and a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule for amplifying the second nucleic acid molecule. Ah When it is determined in step A) that the hybridizing sequence is not present, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are respectively represented by the first tag nucleic acid sequence The primer is designed so as to be included upstream of the 5 ′ end of the reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule, and the hybridizing sequence is determined to be present in the procedure A). The internal hybridizing sequence and the internal hybridizing complementary sequence that hybridizes under the condition that only the specific sequence hybridizes to the internal hybridizing sequence, or the first tag nucleic acid sequence and the second tag. Nucleic acid sequences of the first reverse single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule, respectively. 'So as to include the end upstream, to design primers, including procedures, to provide a recording medium. Here, as described in the above method, each procedure can be performed by designing primers using any known method.

  In another aspect, the present invention is an apparatus for designing two sets of nucleic acid primer sets, and A) compares the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule to determine the first nucleic acid molecule. And nucleic acid sequence determination means for determining whether or not the second nucleic acid molecule has internal hybridizing sequences that hybridize under conditions in which only a specific sequence hybridizes; B) for amplifying the first nucleic acid molecule A first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule, and a second forward one for amplifying the second nucleic acid molecule. A primer design means for designing a second primer set comprising a single-stranded nucleic acid molecule and a second reverse-stranded single-stranded nucleic acid molecule, wherein the hybridizing sequence is determined not to exist in step A) ,Up The first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are respectively represented by 5 of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. 'Primers are designed to be included upstream of the ends, and when it is determined in step A) that the hybridizing sequence is present, only the specific sequence for the internal hybridizing sequence and the internal hybridizing sequence An internal hybridizing complementary sequence that hybridizes under the conditions of hybridizing, or the first tag nucleic acid sequence and the second tag nucleic acid sequence, respectively, the first reverse single-stranded nucleic acid molecule and the second A design device comprising a primer design means for designing a primer so as to be included upstream of the 5 ′ end of a forward single-stranded nucleic acid molecule. To provide. Here, as described in the above method, each means can perform primer design using any known method. Typically, these means can be realized using software in a computer.

  In another aspect, the present invention is an apparatus for synthesizing two sets of nucleic acid primer sets, and A) compares the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule to determine the first nucleic acid molecule. And nucleic acid sequence determination means for determining whether or not the second nucleic acid molecule has internal hybridizing sequences that hybridize under conditions in which only a specific sequence hybridizes; B) for amplifying the first nucleic acid molecule A first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule, and a second forward one for amplifying the second nucleic acid molecule. A primer design means for designing a second primer set comprising a single-stranded nucleic acid molecule and a second reverse-stranded single-stranded nucleic acid molecule, wherein the hybridizing sequence is determined not to exist in step A) ,Up The first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are respectively represented by 5 of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. 'Primers are designed to be included upstream of the ends, and when it is determined in step A) that the hybridizing sequence is present, only the specific sequence for the internal hybridizing sequence and the internal hybridizing sequence An internal hybridizing complementary sequence that hybridizes under the conditions of hybridizing, or the first tag nucleic acid sequence and the second tag nucleic acid sequence, respectively, the first reverse single-stranded nucleic acid molecule and the second A primer design means for designing the primer so as to include upstream of the 5 ′ end of the forward single-stranded nucleic acid molecule; and C) above And means for producing a nucleic acid molecule having the sequence of total primer to provide a synthesizer. Here, A) and B) means can perform primer design using any known method as described in the above method. Typically, these means can be realized using software in a computer. C) Typically, a known oligonucleotide synthesizer (for example, available from Applied Biosystems) can be used as the means.

  In another aspect, the present invention provides a kit for preparing a nucleic acid construct in which a plurality of nucleic acid molecules are linked, and A) two nucleic acid primer sets, wherein the set includes the first nucleic acid molecule. A first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplification to obtain a first double-stranded amplification product; and a second nucleic acid A second primer set comprising a second forward single stranded nucleic acid molecule and a second reverse single stranded nucleic acid molecule for amplifying the molecule to obtain a second double stranded amplification product; The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule include a 3 ′ end portion of the first double-stranded amplification product and the second double-stranded amplification product. The 5 ′ terminal part of is designed to hybridize under conditions where only specific sequences hybridize. Is the two sets of nucleotide primer sets; comprises reagents for the amplification reaction using and B) the primer set, a kit. This primer set can take any form as described herein above. As the reaction reagent used here, any reagent for nucleic acid amplification (for example, PCR reagent; DNA polymerase, buffer, etc.) that is known in the art and can be typically marketed can be adopted. .

  In another aspect, the present invention is a kit for cloning a desired nucleic acid molecule, and A) two nucleic acid primer sets, the set comprising a first nucleic acid molecule amplified by a first A first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule, and a second nucleic acid molecule, to obtain a double-stranded amplification product of A second primer set comprising a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule for obtaining a second double-stranded amplification product, wherein the first order The directional single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule include a 3 ′ end portion of the first double-stranded amplification product and a 5 ′ end portion of the second double-stranded amplification product. Are designed to hybridize under conditions in which only specific sequences hybridize, 2 The nucleotide primer set; includes a and B) said first nucleic acid molecule is a cloning vector, wherein said second nucleic acid molecule is the desired nucleic acid molecule, to provide a kit. Here, the primer set is as described above in the present specification, and any form can be used. As the cloning vector used in this kit, any form can be used, and a vector commercially available from Invitrogen or the like can also be used.

  Accordingly, in another aspect, the present invention provides a method for cloning a desired nucleic acid molecule using a first nucleic acid molecule that is a cloning vector and a second nucleic acid molecule that is the target of cloning. In this method, a nucleic acid construct is produced by linking a plurality of nucleic acid molecules including the first nucleic acid molecule and the second nucleic acid molecule. This cloning method of the present invention comprises (a) a step of amplifying the first nucleic acid molecule to obtain a first double-stranded amplification product; (b) amplifying the second nucleic acid molecule to obtain a second double strand. A step of obtaining a strand amplification product, wherein the 3 ′ end portion of the first double-stranded amplification product and the 5 ′ end portion of the second double-stranded amplification product are hybridized only with a specific sequence. (C) mixing the first and second double-stranded amplification products obtained by steps (a) and (b); A step of denatured into single strands and hybridizing under conditions in which only the specific sequence hybridizes; (d) performing a nucleic acid amplification reaction using the mixture of (c) as a template, A process for obtaining linked nucleic acid amplification products in which nucleic acids have been cloned Encompasses. Here, it is understood that each amplification step and hybridization step can utilize any form that can be employed in the method for producing a nucleic acid construct in which a plurality of nucleic acid molecules are linked as described herein above. Is done.

  In one preferred embodiment, the cloning method of the present invention comprises the following steps: (a) using the first nucleic acid molecule as a template, and hybridizing under nucleic acid amplification conditions to the 3 ′ end portion of the first nucleic acid molecule. First reverse single-stranded nucleic acid molecule that can be used, and first forward single-stranded nucleic acid molecule that can hybridize under the nucleic acid amplification conditions to the 5′-end portion of the complementary strand of the first nucleic acid molecule A step of performing a nucleic acid amplification reaction to obtain a first double-stranded amplification product; (b) using the second nucleic acid molecule as a template, and the nucleic acid at the 3 ′ end portion of the second nucleic acid molecule A second reverse single-stranded nucleic acid molecule that can hybridize under amplification conditions, and a 5 'terminal portion of a complementary strand of the second nucleic acid molecule that can hybridize under the nucleic acid amplification conditions. Using the forward single-stranded nucleic acid molecule of No. 1 as a primer to perform a nucleic acid amplification reaction to obtain a second double-stranded amplification product, wherein the first forward single-stranded nucleic acid molecule and In the second reverse single-stranded nucleic acid molecule, the 3 ′ end portion of the first double-stranded amplification product and the 5 ′ end portion of the second double-stranded amplification product are only specific sequences. Designed to hybridize under hybridizing conditions; (c) mixing the first and second double stranded amplification products obtained by steps (a) and (b); Denatured into a single strand and hybridized under conditions where only specific sequences hybridize; and (d) using the mixture of (c) above as a template and the first reverse single-stranded nucleic acid molecule and The second forward single-stranded nucleic acid molecule as a primer Performing a nucleic acid amplification reaction using Te, comprising the step of obtaining a connecting nucleic acid amplification products in which the desired nucleic acid has been cloned. Here, it is understood that each amplification step and hybridization step can utilize any form that can be employed in the method for producing a nucleic acid construct in which a plurality of nucleic acid molecules are linked as described herein above. Is done.

  In Patent Document 1, there is a drawback that an unnecessary sequence is included in the final nucleic acid fragment, and as such, it has been difficult to successfully use it for cloning. The present invention has been found to produce a complementary primer with the required sequence, and in particular, is considered to be one advantageous advantage of the cloning method of the present invention.

  In one embodiment, the cloning method of the present invention further comprises the following steps: (e) sequencing the ligated nucleic acid amplification product obtained by step (d) to identify the desired nucleic acid. Include.

  In one preferred embodiment, the method of the present invention includes a step of ligating the ligated nucleic acid amplification product obtained by the above step (d) into a circular shape. By circularization, the vector is usually in the form of a plasmid and can be introduced into a suitable host cell. When introduced into a host cell, protein expression or amplification can be easily performed, and selection is facilitated.

  Here, preferably, the circularization step includes phosphorylating the terminal of the ligated nucleic acid amplification product and ligating with ligase. For phosphorylation, an ordinary kinase known in the art can be used, and for ligation, an ordinary ligase known in the art can be utilized.

  In another embodiment, the method of the present invention further comprises the step of introducing the circularized linked nucleic acid amplification product into a host cell and amplifying the linked nucleic acid amplification product. Amplification can be performed under any conditions known in the art. It should be noted that suitable conditions can be readily determined by one skilled in the art once a host cell is provided.

  In a preferred embodiment of the cloning method of the present invention, the reverse single-stranded nucleic acid molecule and the forward single-stranded nucleic acid molecule are sufficient to specifically hybridize and do not interfere with the amplification reaction. Has sequence and length.

  In another embodiment, the cloning method of the present invention is carried out under the condition that the first forward single-stranded nucleic acid molecule hybridizes with the 5 ′ end portion of the second nucleic acid molecule only with the specific sequence. A sequence having a hybridizing sequence, or a sequence in which the second reverse single-stranded nucleic acid molecule hybridizes with the 3 ′ end portion of the first nucleic acid molecule and only the specific sequence. Or both. If both are hybridizing sequences, it is expected that circularization will be simplified.

  In a preferred embodiment of the cloning method of the present invention, the first forward single-stranded nucleic acid molecule has a sequence complementary to the 5 ′ end portion of the second nucleic acid molecule, or the second reverse The directional single-stranded nucleic acid molecule is characterized by having a sequence complementary to the 3 ′ end portion of the first nucleic acid molecule, or both. If both are hybridizing sequences, it is expected that circularization will be simplified.

  In a preferred embodiment of the cloning method of the present invention, the first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are both a first nucleic acid molecule and a second nucleic acid molecule, A tag sequence that does not hybridize under the condition that only the specific sequence hybridizes, and a tag complementary sequence that hybridizes under the condition that only the specific sequence hybridizes to the tag sequence, respectively. .

  In a preferred embodiment of the cloning method of the present invention, the first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are both a first nucleic acid molecule and a second nucleic acid molecule, It includes a tag sequence that does not hybridize under conditions where only the specific sequence hybridizes, and a tag complementary sequence that is complementary to the tag sequence.

  An apparatus for automating part or all of the method of the present invention is also within the scope of the present invention.

  In one embodiment of the invention, at least one of the desired nucleic acid molecules to be ligated has a vector sequence. Suitable vectors for the present invention include plasmids and viruses. When a vector (particularly a cloning vector) is used as one of the nucleic acid molecules, the present invention can be used as a gene cloning technique.

  Examples of the cloning vector include pUC118 / 119 type vector, pUC18 / 19 type vector, pTWV228 / 229 DNA, pBR322 DNA, and the like, which are available from, for example, Roche, TaKaRa and the like.

  In one embodiment of the present invention, a cell into which a nucleic acid construct constructed according to the present invention has been introduced can also be included in the present invention. These cells include bacteria (eg, E. coli), fungi (eg, yeast), animal cells (eg, mammalian cells such as humans, apes, cows, horses, pigs and rodents and non-mammalian cells), Examples include plant cells (eg, angiosperms and gymnosperms). Examples of the method for introducing a nucleic acid construct into a cell include, but are not limited to, transfection, electroporation, and microinjection.

  The nucleic acid construct according to the invention or the peptide encoded thereby can be incorporated into a pharmaceutical composition and administered to a human or other animal. These pharmaceutical compositions comprise oral drugs such as pills, tablets, syrups, capsules, injections or infusions (subcutaneous, intravenous, intraperitoneal, intramuscular, including pharmaceutically acceptable carriers or excipients. And the like, but not limited to, a sterile solution administered by nasal administration, an aerosol administered nasally, a suppository and the like.

  In addition, a method of gene therapy by administering a cell into which a nucleic acid construct according to the present invention has been introduced to a human or other animal can be provided by the present invention.

  The present invention also provides an animal or plant transplanted with a cell into which a nucleic acid construct constructed by the method according to the present invention has been introduced. Examples of a method for using an animal or plant into which a nucleic acid construct according to the present invention has been introduced include, for example, mammals such as cows, horses, sheep and goats that secrete the nucleic acid construct according to the present invention or a peptide encoded by the nucleic acid construct according to the present invention as milk Animals, edible plants (eg moss, fruits) that store nucleic acids constructs according to the invention or peptides encoded by nucleic acid constructs according to the invention or nucleic acid constructs according to the invention in edible parts (eg berries, rhizomes, leaves etc. But not limited to these.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  Hereinafter, although an example explains the composition of the present invention in detail, the present invention is not limited to this. The reagents used in the following were those commercially available from Wako Pure Chemicals, Sigma, Toyobo, New England Biolabs, Amersham, Invtrogen, Funakoshi, Nippon Gene, etc., unless otherwise specified. Although synthetic DNA was commissioned for production by Invitrogen, it is understood that one skilled in the art can synthesize such DNA and RNA using techniques well known in the art.

(Example 1: Specific example of construction method of novel gene construct A + B from gene fragment A and gene fragment B by CTA-PCR method-method using tag-)
(Initial PCR)
Gene fragment A and gene fragment B are amplified separately.

  In order to amplify the gene fragment A, an amplification reaction was carried out using a primer at the 5 'end and a primer at the 3' end (this was a tag sequence bound to the primer).

  First, the double-stranded nucleic acid molecule constituting the gene fragment A was dissociated into single strands by heat.

  Next, the primer set and the single-stranded nucleic acid molecule after dissociation are mixed, and DNA polymerase (for example, KOD-Plus- provided by TOYOBO) is added to the mixture.

  Next, when the amplification reaction is carried out under the following conditions, two types of complementary strands (the same length as the original gene fragment A and a tag added to itself) are synthesized.

This is a two native gene fragment A is 1 molecule, and new gene fragment A + tag sequence T _A.

  Next, for amplification of gene fragment B, a primer at the 5 ′ end (this is a tag sequence that is substantially complementary to the tag sequence used for amplification of gene fragment A bound to the primer. ) And 3 ′ end primers were used for the amplification reaction.

  First, the double-stranded nucleic acid molecule constituting the gene fragment B was dissociated into a single strand by heat.

  The primer set is then mixed with the single-stranded nucleic acid molecule after dissociation, and DNA polymerase (available from TOYOBO) is added to the mixture.

Next, when the amplification reaction is performed under the following conditions, two types of complementary strands (the same length as the original gene fragment B and a tag added to the same) are synthesized.
PCR reaction conditions <br/> Temperature Time
94 ℃ 2 minutes Primary heat denaturation
94 ℃ 15 seconds Thermal denaturation ----- |
55 ℃ 30 seconds Annealing 〜20 cycles
68 ℃ 2 minutes Extension reaction ----- |
68 ° C. 6 minutes final elongation reaction which is two native gene fragment B is one molecule, and new gene fragment B + tag sequence T _B.

Then, after the synthesis reaction, when performing PCR amplification n cycles, new gene fragment A + _{T A} and B + _{T B} is produced in large quantities ⁽² n -1 molecules), and the original gene fragment A or B, 1 It is only molecularly produced. In this way, a large amount of single-stranded nucleic acid molecules constituting the gene fragment to which the tag sequence is bound are synthesized.
Composition of PCR reaction solution
KOD-Plus- (1U / μl) 0.4 μl
10Xbuffer 2 μl
25 mM MgSO ₄ 0.8 μl
2 mMdNTP 2 μl
PCR fragment A 0.5 μl
PCR fragment B 0.5 μl
Water (sterile water) 13.8 μl.

PCR reaction conditions <br/> Temperature Time
94 ° C 3 minutes in heat-denatured ice 1 minute At this time, mix the solution well by pipetting
68 ° C 10 minutes Extension reaction.

  Next, secondary PCR is performed. This is PCR using as a sample a mixture of the products in Step 1, and includes annealing of the tag portion and extension reaction.

  This schematic diagram is shown in FIGS.

When mixed, denatured, and annealed, the following four types of hybrids are formed.
Hybrid I: A + _{T A} double-stranded hybrids II: B + _T double-stranded hybrids III of _B: and A + _{T A} and B + _{T B} is a double-stranded by hybridization with _{T A} and _{T B} in each of the 3 'end hybrid those formed IV: a + _{T a} and B + _{T B,} respectively of 5 'by hybridization with _{T a} and _{T B} at the distal side to form a double-stranded.

  Each of the original gene fragments has one molecule, but these are negligible because they do not participate in hybridization.

Next, when the finished _{annealed, A + T A (→)} / B + T B (←) 1 degrees extension reaction by PCR Te use a primer for amplifying only performed. In this case, among the four types of hybrid, hybrid III _{(ie, A + T A (→)} / B + T B (←)) only becomes the template for PCR of only the following one time.

Step 3. Third PCR
In this reaction, only the hybrid III is the only template for the new gene construct, and only the new gene construct A + B can be purely amplified by performing PCR using the primers P _A (→) and P _B (←).

Composition of PCR reaction solution
KOD-Plus- (1U / μl) 2 μl
10x buffer 10 μl
25 mM MgSO ₄ 4 μl
2 mMdNTP 10 μl
Primer 1 (P _A ((R)): 50 μM) 0.5 μl
Primer 2 (P _B (¬): 50 μM) 0.5 μl
PCR fragment A + B 0.5 μl
72.5 μl of water (sterile water).

PCR reaction conditions <br/> Temperature Time
94 ℃ 2 minutes Primary heat denaturation
94 ℃ 15 seconds Thermal denaturation ----- |
55 ℃ 30 seconds Annealing 〜20 cycles
68 ℃ 4 minutes Extension reaction ----- |
The cycle from heat denaturation to extension reaction is repeated for about 20 cycles.
68 ° C 8 minutes Final extension reaction.

In the CTA-PCR performed in this example, for example, the complementary tag in the CTA-PCR is finally incorporated into a new gene, so that the complementary tag is used using an appropriate sequence inside. Preferably it is designed. Alternatively, mutations can be introduced using such sequences. It seems empirically important that the stage 2 product is thoroughly mixed in the heat denaturation → annealing quenching process. Thus, while not wishing to be bound by theory, mixing during this quenching process seems to be empirically important. Further, it is desirable that the ends of the PCR products are smooth (that is, the double strands have exactly the same length and the ends are aligned). For example, when the DNA polymerase to be used is Taq, since A is added to the end, use of such a polymerase should be avoided. Examples of such polymerases include ACCUZYME DNA Polymerase (Funakoshi), AccuSure DNA Polymerase (Funakoshi), Pwo DNA Polymerase (Roche) Expand ^™ High Fidelity PCR System (Roche), and the like.

  By performing CTA-PCR in the same manner, new gene constructs A + B + C, A + B + C + D, and the like can be similarly produced.

  As described above, the hybrid III shown in FIG. 3 is obtained after the final extension reaction, and the following steps are performed after this tertiary PCR reaction.

(In Hybrid III, one (A or B) is a vector (plasmid))
Purify the final product (Hybrid III) and remove primers etc. ・ Use QIAGEN PCR purification kit (details follow QIAGEN protocol)
↓
Phosphorylation of purified hybrid III terminal (phosphorylation with TAKARA Kinase, according to manufacturer's protocol)
↓
Purification of phosphorylated hybrid III (using QIAGEN PCR purification kit)
↓
Circular ligation of purified hybrid III (ligation using TAKARA ligation kit, according to manufacturer's protocol)
↓
Transformation into E. coli → Hybrid III cloning is complete.

(Example 2: Construction of a novel gene construct A + B in which a downstream region B is bound to a region A of one cDNA of Xenopus laevis)
The following shows an example in which a construct of gene A + B was obtained in which nucleic acid molecule A in the upstream portion of a specific Xenopus cDNA was linked to nucleic acid molecule B in the downstream portion of the cDNA.

  In this example, two fragments (3 ′ end portion of fragment A and 5 ′ end portion of fragment B) obtained by PCR of region A and region B without designing a tag (overlapping portion) on the primer itself were used. The duplicated sequence found in is directly used as a tag.

  Also in this case, exactly the same results can be obtained as in the case of using a primer having a complementary tag sequence inserted so that the overlapping portion has an equivalent length as shown in Example 1.

To create region A fragment, for region A
P _A (→): 5'-TGAACTCGAGCAGCAAATTTACTGCAAACC-3 '(SEQ ID NO: 1)
P _A (←): 3'-ATGGACGACACAAACAGCGAGCAGCAGTT-5 '(SEQ ID NO: 2)
Was used.

Also, to create region B fragment,
P _B (→): 5'-TGGTATCCTCTGAAGATACCATCAGAGAGC-3 '(SEQ ID NO: 3)
P _B (←): 3'-AGTTTGCCATTCAGTCTCTGCTCGAGTGCT-5 '(SEQ ID NO: 4)
Was used.

  The sequence of PCR fragment A (1537b) amplified with primers P (→) and P (←) is shown in FIG. 5 (SEQ ID NO: 5). Note: Bold and underline indicate the primer part used (see FIG. 5).

  The sequence of PCR fragment B (1434b) amplified with primers P (→) and PB (←) is shown in FIG. 6 (SEQ ID NO: 6). Note: Bold and underlined indicate the primer part used, italic part indicates the fragment 67 bp which is an overlapping sequence at the 3 ′ end of A and the 5 ′ end of fragment B is shown (see the lower side of FIG. 6) (SEQ ID NO: 7).

Step 1: Primary PCR (Creation of region A fragment and region B fragment)
Composition of PCR reaction solution
KOD-Plus- (1U / μl) 2 μl
10Xbuffer 10 μl
25 mM MgSO ₄ 4 μl
2 mMdNTP 10 μl
Primer 1 (P _A ((R)) or P _A (¬): 50 μM) 0.5 μl
Primer 2 (P _B (¬) or P _B ((R)): 50 μM) 0.5 μl
Template DNA (fragment A or fragment B: 200 ng / μl) 0.5 μl
72.5 μl of water (sterile water).

PCR reaction conditions <br/> Temperature Time
94 ℃ 2 minutes Primary heat denaturation
94 ℃ 15 seconds Thermal denaturation ----- |
55 ℃ 30 seconds Annealing 〜20 cycles
68 ℃ 2 minutes Extension reaction ----- |
68 ° C 6 min Final extension reaction Here, KOD-Plus- is a product of TOYOBO, buffer (10 times solution), MgSO ₄ solution, dNTP solution is an accessory of KOD-Plus-, and primers are from Invitrogen Specially ordered (same below).

Step 2. Secondary PCR (tag part annealing and extension reaction)
Composition of PCR reaction solution
KOD-Plus- (1U / μl) 0.4 μl
10Xbuffer 2 μl
25 mM MgSO ₄ 0.8 μl
2 mMdNTP 2 μl
PCR fragment A 0.5 μl
PCR fragment B 0.5 μl
Water (sterile water) 13.8 μl.

PCR reaction conditions <br/> Temperature Time
94 ° C 3 minutes in heat-denatured ice 1 minute At this time, mix the solution well by pipetting
68 ° C 10 minutes Extension reaction.

Step 3. Tertiary PCR (preparation of a new construct A + B in which PCR fragment A and PCR fragment B are combined)
Composition of PCR reaction solution
KOD-Plus- (1U / μl) 2 μl
10x buffer 10 μl
25 mM MgSO ₄ 4 μl
2 mMdNTP 10 μl
Primer 1 (P _A ((R)): 50 μM) 0.5 μl
Primer 2 (P _B (¬): 50 μM) 0.5 μl
PCR fragment A + B 0.5 μl
72.5 μl of water (sterile water).

PCR reaction conditions <br/> Temperature Time
94 ℃ 2 minutes Primary heat denaturation
94 ℃ 15 seconds Thermal denaturation ----- |
55 ℃ 30 seconds Annealing 〜20 cycles
68 ℃ 4 minutes Extension reaction ----- |
The cycle from heat denaturation to extension reaction is repeated for about 20 cycles.
68 ° C 8 minutes Final extension reaction.

  The sequence of the finally obtained gene A + B (2,904 bp) is shown in FIG. 7 (SEQ ID NO: 8). Note: Bold and underlined indicate duplicate sequences used.

  Unlike Example 1, in this Example, two fragments obtained by PCR of regions A and B (3 ′ end portion of fragment A and fragment) were not designed with a tag (overlapping part) in the primer itself. The overlapping sequence found in the 5 ′ end part of B) is used as a tag as it is. In this case as well, as shown in the “Summary of the Invention”, exactly the same results can be obtained as in the case of using a primer in which a complementary tag sequence is inserted so that the overlapping portion has an equivalent length.

  In this example, since the nucleic acid molecule A and the nucleic acid molecule B have overlapping portions (regions shown in italics in FIG. 2B), it was not necessary to consider the complementary tag portion in the primer design.

  As described above, the present invention is to connect any two gene fragments without using a restriction enzyme to create a new nucleic acid construct, to clone an arbitrary gene without using a restriction enzyme, Compared with the conventional method using the PCR method using a restriction enzyme, a much simpler and quicker method was provided for the first time. In particular, if a vector is used as the A sequence, it can be used as a novel cloning method.

(Example 3: Cloning example using tag sequence)
Next, an example of cloning by gene amplification using a tag sequence was performed.

(1. Amplification of gene fragments A and B by PCR)
Based on the gene amplification method described in Example 1, a cloning vector was employed on one side, the following reagents were prepared for each fragment, and PCR was performed under the following reaction conditions.
94 ° C 2 minutes
94 ℃ 15 seconds ----- |
57 ° C 30 seconds 15 cycles
68 ℃ 3 minutes ----- |
68 ° C for 6 minutes.

As gene fragment A, plasmid pBluescriptIISK (+) (SEQ ID NO: 9) from STRATAGENE was used.
KOD-Plus- (1U / μl) 0.4μl
10 × buffer 2.0μl
25 mM MgSO ₄ 0.8 μl
2 mM dNTP 2.0 μl
Primer1 (10μM) 0.5μl
Primer2 (10μM) 0.5μl
Gene fragment A (100ng) 1.0μl
12.8 μl of water (sterile water).

The DNA to be cloned (SEQ ID NO: 10) was used as gene fragment B.
KOD-Plus- (1U / μl) 0.4μl
10 × buffer 2.0μl
25 mM MgSO ₄ 0.8 μl
2 mM dNTP 2.0 μl
Primer3 (10μM) 0.5μl
Primer4 (10μM) 0.5μl
Gene fragment B (100ng) 1.0μl
Water (sterile water) 12.8μl
-Extension reaction using a mixture of gene fragments A and B amplified by PCR The following reagents were prepared using the PCR products of gene fragments A and B, and an extension reaction was performed under the following reaction conditions.
94 ° C, 2 minutes, ice-cooled, 1 minute (mix the reaction mixture well by pipetting at this point)
68 ° C for 10 minutes.
KOD-Plus- (1U / μl) 0.4μl
10 × buffer 2.0μl
25 mM MgSO ₄ 0.8 μl
2 mM dNTP 2.0 μl
PCR product (derived from gene fragment A) 0.5 μl
PCR product (derived from gene fragment B) 0.5 μl
13.8 μl of water (sterile water).

The following reagents were prepared using a PCR reaction extension reaction product that amplifies A + B to which gene fragments A and B were linked, and PCR was performed under the following reaction conditions.
94 ° C 2 minutes
94 ℃ 15 seconds ----- |
57 ° C 30 seconds 20 cycles
68 ℃ 3 minutes ----- |
68 ° C for 6 minutes.
KOD-Plus- (1U / μl) 0.4μl
10 × buffer 2.0μl
25 mM MgSO ₄ 0.8 μl
2 mM dNTP 2.0 μl
Extension reaction product 0.5μl
Primer1 (10μM) 0.5μl
Primer4 (10μM) 0.5μl
13.3μl of water (sterile water).

For KOD-Plus-, 10 × buffer, 25 mM MgSO ₄ , and 2 mM dNTP, TOYOBO reagent was used.

Each primer used was synthesized by Invitrogen. pBluescriptII SK (+), full length 2961 bp is shown in FIG. 8 (SEQ ID NO: 9, underlined sequence used for Primer).
Primer1 GGGCTGCAGGAATTCGATATCAAGCTTATC (SEQ ID NO: 11)
Primer2 GGGGGATCCACTAGTTCTAGAGCGG (SEQ ID NO: 12).

The gene fragment B, full length 592 bp, and the nucleotide sequence to be cloned are shown in FIG. 9 (SEQ ID NO: 10, underlined sequence used for Primer). However, only the bolder sequence of Primer3 belongs to gene fragment B (FIG. 8), and the italicized sequence of Primer3 is a tag sequence and is a complementary sequence of Primer2.
Primer3 CCGCTCTAGAACTAGTGGATCCCCCGAAGAAAAACAACTGGAACT (SEQ ID NO: 13)
Primer4 TCTCGAATTCTCAGAGATTAGTGCCGGAAA (SEQ ID NO: 14).

  FIG. 10 shows a schematic diagram of gene fragment A, gene fragment B, and gene fragment tag junction.

(Process after completion of PCR reaction)
Purification of PCR product QIAGEN PCR purification kit was used to purify the PCR product and remove the primers.

Phosphorylation of the purified PCR product end The purified PCR product was phosphorylated at the end of the gene fragment using a TAKARA kinase. The kinase was inactivated by heat denaturation after the kinase treatment.

Ligase reaction of PCR product The phosphorylated gene fragment A + B was ligated in a circular manner using a TAKARA ligation kit.

Transformation The gene fragment A + B (pBluescript II SK (+) into which B was inserted) was transformed into an Escherichia coli strain using a ligase reaction solution using a competent cell of STRATAGENE. When the plasmid was purified based on each transformed single E. coli strain, the gene fragment A + B (pBluescript II SK (+) with B inserted) was confirmed in all plasmids (FIG. 11).

  As described above, it was revealed that the present invention functions as a simple cloning technique that does not use restriction enzymes.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The method provided by the present invention is simpler, faster, more accurate and more cost effective than conventional methods and can be applied to any nucleic acid. In one aspect, the method of the present invention can be used in screening methods that are much simpler and quicker than conventional methods. The method of the present invention can be widely used in fields such as molecular biology, tissue and organ regeneration, regenerative medicine and regenerative medicine, gene therapy, diagnostics, and drug development. The nucleic acid construct constructed by the method of the present invention can be widely used in the production of pharmaceuticals, gene therapy and the like. Therefore, it can be said that the present invention has extremely high industrial applicability.

FIG. 1 is a description of an embodiment of the invention. Request page 3-4. (C) shows the complementary strand synthesis step until (a) is the primary PCR step and (b) is to obtain four types of primary PCR product hybrids. 2A shows the amplification of the sequence of the nucleic acid molecule A of the example, and FIG. 2B shows the amplification of the sequence of the nucleic acid molecule A of the example. FIG. 3 shows specific ligation of nucleic acid ligation in Example 1. FIG. 4 is a computer that executes the program of the present invention. FIG. 5 is a sequence diagram of PCR fragment A (1537b) amplified with primers P (→) and P (←) in Example 2. FIG. 6 is a sequence diagram of PCR fragment B (1434b) amplified in Example 2 using primers P (→) and PB (←). FIG. 7 shows PCR fragment B amplified with primers P (→) and PB (←) in Example 2. FIG. 8 is a sequence diagram of pBluescriptII SK (+) (full length 2961 bp) used in the present invention and primers used for amplification thereof. FIG. 9 is a sequence diagram of the gene fragment B used in the present invention, the full length 592 bp, the base sequence to be cloned, and the primers used to amplify it. FIG. 10 is a sequence diagram of gene fragment A, gene fragment B, and gene fragment tag linking part used in the present invention. FIG. 11 shows the amplification results in Example 3. Lane 1: DNA molecular weight marker; Lane 2: PCR product of gene fragment A (pBluescript II SK (+)), 2961 bp; Lane 3: PCR product of gene fragment B, 592 bp; Lane 4: Linked gene fragment A + B PCR product, 3553 bp.

SEQ ID NO: 1: Forward sequence for preparing region A fragment SEQ ID NO: 2: Reverse sequence for preparing region A fragment SEQ ID NO: 3: Forward sequence for preparing region B fragment SEQ ID NO: 4: Region B fragment Reverse sequence for preparation SEQ ID NO: 5: Sequence of PCR fragment A (1537b) amplified with primers P (→) and P (←) in Example 2 SEQ ID NO: 6: Primer P (→) in Example 2 and SEQ ID NO: 7 of PCR fragment B1434b) amplified with PB (←): 67 bp SEQ ID NO: 8 which is an overlapping sequence at the 3 ′ end of fragment A and the 5 ′ end of fragment B: final in Example 2 SEQ ID NO: 9 of the gene A + B (2,904 bp) thus obtained: SEQ ID NO: 10 of the plasmid pBluescriptII SK (+): the desired sequence used in Example 3 SEQ ID NO: 11: used in Example 3 Primer 1
SEQ ID NO: 12: Primer 2 used in Example 3
SEQ ID NO: 13: Primer 3 used in Example 3
SEQ ID NO: 14: Primer 4 used in Example 3

Claims (35)

A method for producing a nucleic acid construct in which a plurality of nucleic acid molecules including a first nucleic acid molecule and a second nucleic acid molecule are linked, the method comprising:
(A) amplifying the first nucleic acid molecule to obtain a first double-stranded amplification product;
(B) a step of amplifying a second nucleic acid molecule to obtain a second double-stranded amplification product, wherein the 3 ′ end portion of the first double-stranded amplification product and the second double-stranded product Amplifying the 5 ′ end portion of the amplification product to have a sequence that hybridizes under conditions in which only specific sequences hybridize;
(C) The first and second double-stranded amplification products obtained by steps (a) and (b) are mixed, denatured into single strands, and only the specific sequence hybridizes. And (d) performing a nucleic acid amplification reaction using the mixture of (c) as a template to obtain a linked nucleic acid amplification product. The method of claim 1, wherein the method comprises:
(A) using the first nucleic acid molecule as a template, a first reverse single-stranded nucleic acid molecule capable of hybridizing to the 3 ′ end portion of the first nucleic acid molecule under nucleic acid amplification conditions, and the first Using the first forward single-stranded nucleic acid molecule hybridizable under the nucleic acid amplification conditions as a primer to the 5 ′ end portion of the complementary strand of the nucleic acid molecule, a nucleic acid amplification reaction is performed to obtain a first double-stranded amplification product Obtaining
(B) using the second nucleic acid molecule as a template, a second reverse single-stranded nucleic acid molecule capable of hybridizing to the 3 ′ end portion of the second nucleic acid molecule under the nucleic acid amplification conditions, and the second A second double-stranded amplification by performing a nucleic acid amplification reaction using as a primer a second forward single-stranded nucleic acid molecule capable of hybridizing under the nucleic acid amplification conditions to the 5 ′ end portion of the complementary strand of the nucleic acid molecule of Obtaining a product, wherein the first forward single stranded nucleic acid molecule and the second reverse single stranded nucleic acid molecule comprise a 3 ′ end portion of the first double stranded amplification product, And the 5 ′ end portion of the second double-stranded amplification product are designed to hybridize under conditions where only specific sequences hybridize;
(C) The first and second double-stranded amplification products obtained in steps (a) and (b) are mixed, denatured into single strands, and under conditions where only specific sequences hybridize. And (d) using the mixture of (c) as a template, and using the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule as primers. Performing a nucleic acid amplification reaction to obtain a linked nucleic acid amplification product. The method of claim 1, wherein:
(E) amplifying the double-stranded nucleic acid molecule obtained by step (d),
Further comprising a method.   The reverse single-stranded nucleic acid molecule and the forward single-stranded nucleic acid molecule have a sequence and length sufficient to specifically hybridize and do not interfere with the amplification reaction. The method described.   The first forward single-stranded nucleic acid molecule has a sequence that hybridizes with the 5 ′ end portion of the second nucleic acid molecule under conditions in which only the specific sequence hybridizes, or the second The reverse-direction single-stranded nucleic acid molecule has a sequence that hybridizes with the 3 ′ end portion of the first nucleic acid molecule and a condition that only the specific sequence hybridizes, or both. The method according to claim 2.   The first forward single stranded nucleic acid molecule has a sequence complementary to the 5 ′ end portion of the second nucleic acid molecule, or the second reverse single stranded nucleic acid molecule is the first 6. A method according to claim 5, characterized in that it has a sequence complementary to the 3 'end part of the nucleic acid molecule or both.   Conditions under which the first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are hybridized only with the specific sequence in both the first nucleic acid molecule and the second nucleic acid molecule. The method according to claim 2, further comprising: a tag sequence that does not hybridize with the tag sequence; and a tag complementary sequence that hybridizes under conditions where only the specific sequence hybridizes to the tag sequence.   Conditions under which the first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are hybridized only with the specific sequence in both the first nucleic acid molecule and the second nucleic acid molecule. The method according to claim 7, comprising a tag sequence that does not hybridize with the tag sequence and the tag complementary sequence that is complementary to the tag sequence. The method of claim 1, wherein at least one of the first nucleic acid molecule or the second nucleic acid molecule has at least part or all of the sequence of the vector. The method according to claim 9, wherein the vector is a cloning vector. Amplification of the nucleic acid includes polymerase chain reaction (PCR), ligase chain reaction (LCR), gap LCR, strand displacement amplification (SDA), Qβ replicase amplification, transcription amplification system (TAS), self-supporting sequence replication system (Self-sustained sequence replication system; 3SR); nucleic acid sequence-based amplification (NASBA); transcription-mediated amplification (TMA), chimeric primer-initiated nucleic acid amplification (ICAN) and loop-mediated isothermal amplification (LAMP) under isothermal conditions The method of claim 1, wherein the method is performed by a selected amplification method. The method of claim 1, wherein the nucleic acid comprises DNA. A nucleic acid construct obtained by the method according to claim 1. Two nucleic acid primer sets, the set comprising a first forward single stranded nucleic acid molecule and a first nucleic acid molecule for amplifying the first nucleic acid molecule to obtain a first double stranded amplification product; A second forward single stranded nucleic acid molecule and a first primer set for amplifying the second nucleic acid molecule to obtain a second double stranded amplification product, the first primer set comprising a reverse single stranded nucleic acid molecule; A second primer set comprising two reverse single stranded nucleic acid molecules,
The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule include a 3 ′ end portion of the first double-stranded amplification product and the second double-stranded amplification product. Is designed to hybridize under conditions in which only specific sequences hybridize,
Two nucleic acid primer sets. A method for designing two nucleic acid primer sets,
A) The inside where the first nucleic acid molecule and the second nucleic acid molecule are hybridized under the condition that only the specific sequence hybridizes by comparing the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule. Determining whether they have hybridizing sequences to each other;
B) a first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule; and the second nucleic acid molecule Designing a second primer set comprising a second forward single stranded nucleic acid molecule and a second reverse single stranded nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing conditions are respectively converted into the first reverse sequence. A primer is designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule;
In step A), when it is determined that the hybridizing sequence is present, the internal hybridizing sequence is performed so that only the specific sequence hybridizes with the internal hybridizing sequence and the internal hybridizing sequence. Complementary sequences, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. Designing a primer to include a process,
Including the method. A method for producing two nucleic acid primer sets comprising:
A) The inside where the first nucleic acid molecule and the second nucleic acid molecule are hybridized under the condition that only the specific sequence hybridizes by comparing the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule. Determining whether they have hybridizing sequences to each other;
B) a first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule; and the second nucleic acid molecule Designing a second primer set comprising a second forward single stranded nucleic acid molecule and a second reverse single stranded nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing conditions are respectively converted into the first reverse sequence. A primer is designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule;
In step A), when it is determined that the hybridizing sequence is present, the internal hybridizing sequence is performed so that only the specific sequence hybridizes with the internal hybridizing sequence and the internal hybridizing sequence. Complementary sequences, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. Designing a primer to include; and C) producing a nucleic acid molecule having the sequence of the designed primer. A program for designing two nucleic acid primer sets,
A) Conditions for allowing the nucleic acid sequence determination means to compare the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule so that only the specific sequences of the first nucleic acid molecule and the second nucleic acid molecule hybridize. Determining whether they have internal hybridizing sequences that hybridize under each other;
B) a first primer set including a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule in the primer designing means; A procedure for designing a second primer set comprising a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule for amplifying a second nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are respectively converted into the first reverse sequence. A primer is designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule;
In the step A), when it is determined that the hybridizing sequence is present, the internal hybridizing sequence is performed so that only the specific sequence hybridizes with the internal hybridizing sequence and the internal hybridizing sequence. Complementary sequences, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. Let the primers be designed to include procedures,
Including the program. A recording medium storing a program for designing two nucleic acid primer sets, the program comprising:
A) Conditions for allowing the nucleic acid sequence determination means to compare the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule so that only the specific sequences of the first nucleic acid molecule and the second nucleic acid molecule hybridize. Determining whether they have internal hybridizing sequences that hybridize under each other;
B) a first primer set including a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule in the primer designing means; A procedure for designing a second primer set comprising a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule for amplifying a second nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are respectively converted into the first reverse sequence. A primer is designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule;
In the step A), when it is determined that the hybridizing sequence is present, the internal hybridizing sequence is performed so that only the specific sequence hybridizes with the internal hybridizing sequence and the internal hybridizing sequence. Complementary sequences, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. Let the primers be designed to include procedures,
Including a recording medium. A device for designing two nucleic acid primer sets,
A) The inside where the first nucleic acid molecule and the second nucleic acid molecule are hybridized under the condition that only the specific sequence hybridizes by comparing the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule. Nucleic acid sequence determination means for determining whether or not to have hybridizing sequences with each other;
B) a first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule; and the second nucleic acid molecule A primer design means for designing a second primer set comprising a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are respectively converted into the first reverse sequence. A primer is designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule;
In the step A), when it is determined that the hybridizing sequence is present, the internal hybridizing sequence is performed so that only the specific sequence hybridizes with the internal hybridizing sequence and the internal hybridizing sequence. Complementary sequences, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. A design apparatus comprising primer design means for designing a primer to include. An apparatus for synthesizing two nucleic acid primer sets,
A) The inside where the first nucleic acid molecule and the second nucleic acid molecule are hybridized under the condition that only the specific sequence hybridizes by comparing the nucleic acid sequences of the first nucleic acid molecule and the second nucleic acid molecule. Nucleic acid sequence determination means for determining whether or not to have hybridizing sequences with each other;
B) a first primer set comprising a first forward single-stranded nucleic acid molecule and a first reverse single-stranded nucleic acid molecule for amplifying the first nucleic acid molecule; and the second nucleic acid molecule A primer design means for designing a second primer set comprising a second forward single-stranded nucleic acid molecule and a second reverse single-stranded nucleic acid molecule,
In the step A), when it is determined that the hybridizing sequence does not exist, the first tag nucleic acid sequence and the second tag nucleic acid sequence that hybridize with each other under the hybridizing condition are respectively converted into the first reverse sequence. A primer is designed to be included upstream of the 5 ′ end of the directional single stranded nucleic acid molecule and the second forward single stranded nucleic acid molecule;
In the step A), when it is determined that the hybridizing sequence is present, the internal hybridizing sequence is performed so that only the specific sequence hybridizes with the internal hybridizing sequence and the internal hybridizing sequence. Complementary sequences, or the first tag nucleic acid sequence and the second tag nucleic acid sequence are respectively upstream of the 5 ′ end of the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule. A synthesis apparatus comprising: a primer design means for designing a primer to include; and C) a means for producing a nucleic acid molecule having the sequence of the designed primer. A kit for producing a nucleic acid construct in which a plurality of nucleic acid molecules are linked,
A) Two nucleic acid primer sets, the set comprising a first forward single-stranded nucleic acid molecule and a first nucleic acid molecule for amplifying the first nucleic acid molecule to obtain a first double-stranded amplification product. A first forward single-stranded nucleic acid molecule for amplifying the second nucleic acid molecule to obtain a second double-stranded amplification product by comprising a first primer set comprising one reverse single-stranded nucleic acid molecule And a second primer set comprising a second reverse single stranded nucleic acid molecule,
The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule include a 3 ′ end portion of the first double-stranded amplification product and the second double-stranded amplification product. Is designed to hybridize under conditions in which only specific sequences hybridize,
A kit comprising two nucleic acid primer sets; and B) a reaction reagent for an amplification reaction using the primer set. A kit for cloning a desired nucleic acid molecule,
A) Two nucleic acid primer sets, the set comprising a first forward single-stranded nucleic acid molecule and a first nucleic acid molecule for amplifying the first nucleic acid molecule to obtain a first double-stranded amplification product. A first forward single-stranded nucleic acid molecule for amplifying the second nucleic acid molecule to obtain a second double-stranded amplification product by comprising a first primer set comprising one reverse single-stranded nucleic acid molecule And a second primer set comprising a second reverse single stranded nucleic acid molecule,
The first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule include a 3 ′ end portion of the first double-stranded amplification product and the second double-stranded amplification product. Is designed to hybridize under conditions in which only specific sequences hybridize,
Two nucleic acid primer sets; and B) the first nucleic acid molecule being a cloning vector;
Wherein the second nucleic acid molecule is the desired nucleic acid molecule. A method for cloning a desired nucleic acid molecule using a first nucleic acid molecule that is a cloning vector and a second nucleic acid molecule that is a target for cloning, the method comprising: A nucleic acid construct in which a plurality of nucleic acid molecules including a second nucleic acid molecule are linked is prepared, and the method includes the following:
(A) amplifying the first nucleic acid molecule to obtain a first double-stranded amplification product;
(B) a step of amplifying the second nucleic acid molecule to obtain a second double-stranded amplification product, the 3 ′ end portion of the first double-stranded amplification product, and the second double-stranded amplification product A step wherein the 5 ′ end portion of the strand amplification product is amplified to have a sequence that hybridizes under conditions in which only specific sequences hybridize;
(C) The first and second double-stranded amplification products obtained by steps (a) and (b) are mixed, denatured into single strands, and only the specific sequence hybridizes. Hybridizing with:
(D) A step of performing a nucleic acid amplification reaction using the mixture of (c) as a template to obtain a linked nucleic acid amplification product in which the desired nucleic acid is cloned. 24. The method of claim 23, comprising:
(A) using the first nucleic acid molecule as a template, a first reverse single-stranded nucleic acid molecule capable of hybridizing to the 3 ′ end portion of the first nucleic acid molecule under nucleic acid amplification conditions, and the first Using the first forward single-stranded nucleic acid molecule hybridizable under the nucleic acid amplification conditions as a primer to the 5 ′ end portion of the complementary strand of the nucleic acid molecule, a nucleic acid amplification reaction is performed to obtain a first double-stranded amplification product Obtaining
(B) using the second nucleic acid molecule as a template, a second reverse single-stranded nucleic acid molecule capable of hybridizing to the 3 ′ end portion of the second nucleic acid molecule under the nucleic acid amplification conditions, and the second A second double-stranded amplification by performing a nucleic acid amplification reaction using as a primer a second forward single-stranded nucleic acid molecule capable of hybridizing under the nucleic acid amplification conditions to the 5 ′ end portion of the complementary strand of the nucleic acid molecule of Obtaining a product, wherein the first forward single stranded nucleic acid molecule and the second reverse single stranded nucleic acid molecule comprise a 3 ′ end portion of the first double stranded amplification product, And the 5 ′ end portion of the second double-stranded amplification product are designed to hybridize under conditions where only specific sequences hybridize;
(C) The first and second double-stranded amplification products obtained in steps (a) and (b) are mixed, denatured into single strands, and under conditions where only specific sequences hybridize. And (d) using the mixture of (c) as a template, and using the first reverse single-stranded nucleic acid molecule and the second forward single-stranded nucleic acid molecule as primers. A method comprising a step of performing a nucleic acid amplification reaction to obtain a linked nucleic acid amplification product in which the desired nucleic acid is cloned. 24. The method of claim 23, wherein:
(E) sequencing the ligated nucleic acid amplification product obtained in step (d) to identify the desired nucleic acid;
Further comprising a method.   The method according to claim 23, comprising the step of ligating the ligated nucleic acid amplification product obtained by step (d) into a circle. 27. The method according to claim 26, wherein the step of circularizing comprises phosphorylating an end of the ligated nucleic acid amplification product and ligating with ligase. 27. The method of claim 26, further comprising the step of introducing the circularized linked nucleic acid amplification product into a host cell and amplifying the linked nucleic acid amplification product.   25. The reverse single stranded nucleic acid molecule and the forward single stranded nucleic acid molecule have a sequence and length sufficient to specifically hybridize and not interfere with the amplification reaction. The method described.   The first forward single-stranded nucleic acid molecule has a sequence that hybridizes with the 5 ′ end portion of the second nucleic acid molecule under conditions in which only the specific sequence hybridizes, or the second The reverse-direction single-stranded nucleic acid molecule has a sequence that hybridizes with the 3 ′ end portion of the first nucleic acid molecule and a condition that only the specific sequence hybridizes, or both. 25. The method of claim 24.   The first forward single stranded nucleic acid molecule has a sequence complementary to the 5 ′ end portion of the second nucleic acid molecule, or the second reverse single stranded nucleic acid molecule is the first The method according to claim 30, characterized in that it has a sequence complementary to the 3 'terminal part of the nucleic acid molecule or both.   Conditions under which the first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are hybridized only with the specific sequence in both the first nucleic acid molecule and the second nucleic acid molecule. 25. The method according to claim 24, comprising: a tag sequence that does not hybridize with the tag sequence; and a tag complementary sequence that hybridizes under conditions where only the specific sequence hybridizes to the tag sequence.   Conditions under which the first forward single-stranded nucleic acid molecule and the second reverse single-stranded nucleic acid molecule are hybridized only with the specific sequence in both the first nucleic acid molecule and the second nucleic acid molecule. 33. The method according to claim 32, comprising a tag sequence that does not hybridize with said tag sequence and said tag complementary sequence that is complementary to said tag sequence. A cell comprising a nucleic acid construct obtained by the method according to claim 1. A polypeptide encoded by a nucleic acid construct obtained by the method of claim 1.