JP2011010591A - Method for amplifying dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a practical method for amplifying a DNA.SOLUTION: The method for amplifying the DNA includes treating the DNA with one or more kinds of restriction enzymes forming an overhang having a length of five or more bases and containing one or more arbitrary base sites when amplifying the DNA to prepare a first double-stranded DNA fragment, linking to the first double-stranded DNA fragment, an adaptor DNA having a linkage sequence containing a base sequence complementary to the base sequence of the overhang and an amplification sequence containing a common base sequence for the amplification to acquire a second double-stranded DNA fragment, and amplifying the second double-stranded DNA fragment by using a DNA polymerase.

Description

  The present invention relates to a method for amplifying DNA, and more particularly, to a method for amplifying arbitrary DNA such as genomic DNA and use thereof.

One method for amplifying DNA is the whole genome amplification (WGA) method. The WGA method is considered to be a useful means for amplifying DNA to about 10 ⁴ to 10 ⁵ and obtaining a large amount of DNA from a rare DNA sample. It is a useful tool for various biomedical applications such as microsatellite analysis, single nucleotide polymorphism (SNP) detection, and comparative genomic hybridization (CGH) microarrays. As the WGA method, an LMP (Ligation-Mediated PCR) method and an MDA (Multiple Displacement Amplification) method are known.

  The MDA method is a method for amplifying genomic DNA using bacteriophage-derived phi29 DNA polymerase (Non-patent Document 1). Random hexamer primer is annealed to template genomic DNA, complementary strand synthesis is started by phi29 DNA polymerase, primer is annealed to the synthesized complementary strand, and synthesis by phi29 DNA polymerase is started to continuously amplify DNA Is done. In the LMP method, genomic DNA is made into fragments of about 500 bp, and then a library is created by adding adapters as primers to both ends of the fragments. It is possible (Non-Patent Document 2).

Dean F.B. et al., Proc. Natl. Acad. Sci. USA 99 (8), 5261-5266 (2002) Klein C.A. et al., Proc. Natl. Acad. Sci. USA 96 (8), 4494-4499 (1999)

  However, since all the MDA methods are difficult to amplify DNA uniformly, there is a possibility that DNA cannot be analyzed accurately. In addition, the LMP method has a problem that side reactions are likely to occur when the efficiency of ligation is not high because DNA is mainly cleaved with Dnase I and ligation is performed using the blunt ends. Furthermore, it is necessary to control the length of the cleaved fragment by strictly controlling the amount of enzyme and the processing time to realize PCR at a constant temperature cycle. However, in practice, such control is extremely difficult. It was.

  From the above, at present, a more practical DNA amplification method is required. Accordingly, it is an object of the present invention to provide a more practical DNA amplification method.

  When amplifying DNA, the present inventors cut the DNA to be amplified with a restriction enzyme so that an overhang containing a certain base length and containing an arbitrary base is generated, and an amplification step is performed using this DNA fragment. As a result, it was found that the DNA to be amplified can be amplified uniformly and efficiently, and the present invention was completed. That is, according to the present invention, the following means are provided.

  According to the disclosure of the present specification, it is a method for amplifying DNA, and has one or two or more restriction enzymes that form an overhang having a base length of 5 bases or more and including one or more arbitrary base sites. A step of preparing the first double-stranded DNA fragment by treating the DNA with a linking sequence comprising a base sequence complementary to the overhanging base sequence in the first double-stranded DNA fragment; A step of linking an adapter DNA having an amplification sequence including a common base sequence for amplification to obtain a second double-stranded DNA fragment; and the second double-stranded DNA fragment is converted into a DNA polymerase. And amplifying using the method.

  In this amplification method, the step of obtaining the first double-stranded DNA fragment can include treatment with the restriction enzyme that forms the overhang having a base length of 9 bases. In this embodiment, the restriction enzyme can be TspRI.

  In the present amplification method, the step of obtaining the second double-stranded DNA fragment has the amplification sequence including the base sequence common to the 3 ′ end side and the 5 ′ end side of the double-stranded DNA fragment. The step can be performed using adapter DNA. In this embodiment, the amplification step may be a step of amplifying the second DNA fragment by PCR using a primer complementary to the amplification sequence.

  In the present amplification method, the step of obtaining the second double-stranded DNA fragment includes a base sequence capable of forming a terminal hairpin structure on each of the 3 ′ end side and the 5 ′ end side of the double-stranded DNA fragment. The adapter DNA having the amplification sequence can be used as a step. In this embodiment, the amplification step can be a step of amplifying the second double-stranded DNA by isothermal amplification.

  In the present amplification method, the step of preparing the first double-stranded DNA fragment may be a step of preparing the first double-stranded DNA fragment by treating the DNA with one kind of the restriction enzymes. it can. The step of preparing the first double-stranded DNA fragment is a step of preparing the second double-stranded DNA fragment by treating the DNA with two or more kinds of the restriction enzymes, respectively. The second double-stranded DNA fragment obtaining step obtains the second double-stranded DNA fragment for each of the first double-stranded DNA fragments obtained for two or more kinds of the restriction enzymes. The amplification step may be a step of performing an amplification step on the second double-stranded DNA fragment obtained for each of two or more kinds of restriction enzymes.

  According to the disclosure of the present specification, a kit for amplifying DNA, which has a base length of 5 bases or more and forms an overhang containing one or more arbitrary base sites, is one or two or more restriction enzymes And an adapter DNA having a linking sequence that includes a base sequence complementary to the base sequence of the overhang and an amplification sequence that includes a common base sequence for amplification. .

  The kit may further include a primer having a base sequence complementary to a common amplification sequence in the adapter DNA. In the kit, the adapter DNA may have the amplification sequence including a base sequence capable of forming a terminal hairpin structure.

It is a figure which shows an example of the outline | summary of this invention. It is a figure which shows the other aspect (constant temperature amplification process) of an amplification process. In Example 1, it is a figure which shows the electrophoresis analysis result of the amplified DNA. FIG. 3 (a) shows the composition of the fragment of plasmid pQBI T7-GFP DNA digested with TspRI, and FIG. 3 (b) shows an electrophoretogram of the amplified DNA. It is a figure which shows the electrophoretic analysis result of the whole genomic DNA of E.coli K-12 amplified in Example 2. Lane 1 shows the results of DNA amplification from several tens of E. coli K-12, and lane 2 shows a control. It is a figure which shows the electrophoretic analysis result of the total genomic DNA of the human cell amplified in Example 3. Lane 1 shows the results of DNA amplification from about 100 human 293FT cells, Lane 2 shows the results of DNA amplification from about 10 human 293T cells, and Lane 3 shows the results from about 1 human 293T cell. The result of DNA amplification is shown.

  The method for amplifying DNA disclosed in the present specification is a method for amplifying the DNA with one or two or more restriction enzymes having a base length of 5 bases or more and forming an overhang containing one or more arbitrary base sites. Processing to prepare a first double-stranded DNA fragment, a linking sequence containing a base sequence complementary to the base sequence of the overhang in the first double-stranded DNA fragment, and amplification A step of obtaining a second double-stranded DNA fragment by ligating an adapter DNA having a common amplification sequence containing a base sequence, and amplifying the second double-stranded DNA fragment using a DNA polymerase; And a step of performing.

  According to this amplification method, by treating with a specific restriction enzyme, a first double-stranded DNA fragment having an overhang having both ease of annealing and specificity can be prepared. Since this first double-stranded DNA fragment has an appropriate overhang, it is efficiently and specifically annealed with the adapter DNA and ligated to the second double-stranded DNA having a common amplification sequence. DNA fragments can be obtained efficiently and accurately. Furthermore, the amplification process can be carried out easily and efficiently by amplifying the second double-stranded DNA fragment using a common amplification sequence.

  That is, according to the present amplification method, the first double-stranded DNA fragment is prepared using a restriction enzyme capable of forming an overhang of a predetermined form, thereby obtaining the second double-stranded DNA fragment in the obtaining step. In addition to ensuring specificity and efficiency, it is possible to ensure the efficiency and homogeneity of the amplification process. In this way, it is not found before the filing date of the present application that the high efficiency and good amplification can be easily achieved by utilizing the diversity of overhangs.

  Hereinafter, various embodiments of such an amplification method and use thereof will be described. FIG. 1 is a diagram illustrating an outline of an example of the amplification method disclosed in this specification.

(DNA amplification method)
The DNA amplification method disclosed in the present specification is a method for amplifying DNA in a sample, and is not intended to specifically amplify specific DNA in a sample or a specific region in DNA. Preferably, the amplification method is intended to amplify the DNA in the sample as non-specifically as uniformly as possible. Moreover, it can be said that this amplification method is a method for producing a replica of DNA in a sample. Here, the replica means that the DNA in the sample is replicated by fragmenting into a plurality of double-stranded DNA fragments.

  This amplification method can also be used as a method for preparing a library suitable for amplification. According to the amplification method disclosed in the present specification, a library can be efficiently constructed from DNA.

(DNA to be amplified)
The DNA to be amplified by the amplification method disclosed herein is not particularly limited. It may be the genomic DNA of humans or viruses, including humans, or a part thereof, or may be a non-living DNA such as prion or a part thereof, or a DNA enzymatically synthesized using RNA. It may be a chemically synthesized DNA. The DNA to be amplified may contain multiple types of DNA. Further, the form may be a chain, a ring, or a mixture thereof. The direct amplification target is preferably double-stranded DNA. When single-stranded DNA or RNA is to be amplified, it is preferable to appropriately prepare double-stranded DNA by a known method. The sample containing the DNA to be amplified is not particularly limited. It may contain body fluids, excreta, tissues and cells or extracts thereof collected from living organisms such as humans, and may be foods or extracts thereof. It may be a proliferation such as a microorganism or a virus.

(Step of preparing the first double-stranded DNA fragment)
In this step, the first double-stranded DNA fragment is obtained by treating DNA with one or more restriction enzymes having a base length of 5 bases or more and forming an overhang containing one or more arbitrary base sites. It can be set as the process of preparing. By carrying out this step, a double-stranded DNA fragment having an overhang having a base length of 5 bases or more and containing one or more arbitrary base sites can be prepared. Since the first double-stranded DNA fragment has such an overhang, the second step of preparing the second double-stranded DNA fragment can be accurately and efficiently performed.

  In this step, a DNA to be amplified is cleaved using a restriction enzyme that forms an overhang to prepare a first double-stranded DNA fragment having an overhang. As the restriction enzyme, a restriction enzyme that forms an overhang having a base length of 5 bases or more can be used. When the overhang is 5 bases or more, the linking specificity with the adapter DNA can be ensured in the subsequent step, so that the second double-stranded DNA can be obtained with certainty. The overhang formed by the restriction enzyme is more preferably 6 bases or more, still more preferably 7 bases or more, still more preferably 8 bases or more, and most preferably 9 bases or more.

The overhang generated by the restriction enzyme preferably contains an arbitrary base site of one base or two or more bases. In the present specification, the arbitrary base portion refers to a portion to which any of two or more bases of A, T, G and C are arbitrarily applied. Therefore, in addition to the site which may be any of A, T, G and C, the site which may be C or G is also included in the arbitrary base site. As already explained, by including an arbitrary base site, dimer formation due to annealing between overhangs can be suppressed based on palindromic sequences between overhangs, and complementation with adapter DNA described later Can be efficiently and accurately connected. Arbitrary base sites of 2 bases or more may be continuous, or may be arranged via bases constituting a recognition site of 1 base or 2 bases or more included in the overhang. The arbitrary base is preferably 3 bases or more, more preferably 4 bases or more. For example, in the case of Hga I, the overhang of 5 bases is NNNNN, and if 1024 types (4 ⁵ ) of adapter DNAs are used, all sequences can be amplified. Therefore, when treated with Hga I, the overhang formed in the first double-stranded DNA fragment has a sequence consisting of five consecutive bases specific to the double-stranded DNA fragment. By using an adapter DNA complementary to such a specific overhang, the homogeneity of amplification of the target DNA is improved.

  The number of bases constituting the recognition site of the restriction enzyme is not particularly limited, but can be determined in consideration of whether or not the length of the first double-stranded DNA fragment to be prepared is suitable for amplification. When the number of bases constituting the recognition site is large, the presence frequency of the recognition site is low, and on average, a longer first double-stranded DNA fragment is formed. When the number of bases is small, the recognition site The frequency of existence becomes higher, and on average, shorter first double-stranded DNA fragments are formed. For example, when the number of bases constituting the recognition site is 4 to 6 bases (typically 5 bases), a double-stranded DNA fragment having a preferred length can be obtained from the relationship of amplification efficiency and the like. For example, in the case of TspRI, if there is a sequence of CASTG (CACTG or CAGTG), it can be cleaved. On the average, the cleavage site of TspRI is considered to be one place in about 400-500 bases. The recognition site may be in an overhang or may be other than an overhang.

  Examples of such restriction enzymes include Hga I (5-base overhang, 5-base recognition sequence), Bae I (5-base overhang, 6.5-base recognition sequence), Hin4 I (5-base overhang). 5 base recognition sequence), Alo I (5 base overhang, 7 base recognition sequence), Bsp24 I (5 base overhang, 6 base recognition sequence), Hae IV (5 base and 6 base overhang) Hang, 5 base recognition sequence), Cje I (6 base overhang, 5 base recognition sequence), CjeP I (6 base overhang, 5 base recognition sequence) and TspRI (9 base overhang, 4 .5 base recognition sequence). Among them, a restriction enzyme that generates an overhang of 6 bases or more is preferable, and a restriction enzyme that generates an overhang of 9 bases is more preferable.

  Restriction enzymes can be used alone or in combination of two or more. That is, the first double-stranded DNA fragment may be prepared by allowing only one type of restriction enzyme to act on the DNA to be amplified, or two or more types of restriction enzymes may act simultaneously on DNA. You may let them. By using one type of restriction enzyme, the adapter can be prepared according to the overhang formed by the one type of restriction enzyme, thus simplifying the preparation process of the first double-stranded DNA fragment. be able to. In addition, by simultaneously acting two or more types of restriction enzymes, for example, even when a restriction enzyme having a recognition sequence having a long base number is used, a first double-stranded DNA fragment having an appropriate length can be obtained. . When two or more types of restriction enzymes are used, it is necessary to facilitate the type of adapter DNA according to each overhang formed by two or more types of restriction enzymes.

  Two or more kinds of restriction enzymes may be allowed to act on DNA. That is, the step of preparing the first double-stranded DNA fragment for each restriction enzyme may be performed by cleaving DNA with different restriction enzymes. In this way, it is difficult to avoid the generation of fragments that cannot be ligated to adapter DNA with only one type of restriction enzyme, and there is a possibility that a DNA region that cannot be amplified may be generated. By forming different sets of first double-stranded DNA fragments at different cleavage sites, the number of DNA regions that can be avoided from amplification can be reduced as much as possible.

  As described above, the first double-stranded DNA fragment having an overhang that can be generated by the used restriction enzyme can be prepared by cleaving the amplification target DNA using the restriction enzyme. Even when one type of restriction enzyme is used, since the overhang has an arbitrary base site of one or more bases, the mode of the overhang is not necessarily limited to one type of base sequence, and there are a plurality of types of modes. Further, in this step, usually, a plurality of first double-stranded DNA fragments are generated.

(Second double-stranded DNA fragment acquisition step)
In this step, the first double-stranded DNA fragment has one or two ligation sequences including a base sequence complementary to the overhang base sequence and an amplification sequence including a common base sequence for amplification. It is possible to obtain a second double-stranded DNA fragment by linking adapter DNAs of more than two species. According to this step, the first double-stranded DNA fragment can be prepared in a form suitable for amplification. By including a common amplification sequence, efficient amplification is possible.

(Adapter DNA)
The adapter DNA used in this step has at least a linking sequence containing a base sequence complementary to the overhang of the first double-stranded DNA fragment and an amplification sequence containing a common base sequence for amplification. If it is, it is enough. The sequence for ligation can be determined based on the base sequence of the overhang that will be formed by the restriction enzyme used for the preparation of the first double-stranded DNA fragment. That is, a linking sequence including a base sequence complementary to all of the possible base sequences is determined and prepared so that it can be linked to a base sequence that may cause overhang. In this way, all the first double-stranded DNA fragments that can be generated by the restriction enzyme treatment can be surely prepared as second double-stranded DNA fragments. For this purpose, a base sequence in which four types of bases A, T, G and C are arranged for each arbitrary base site included in the overhang is designed. For example, when there is one arbitrary base site in the overhang, an adapter DNA having four types of ligation sequences each having 4 bases introduced into the one arbitrary base site is prepared and prepared. There is a need.

  Further, when the adapter DNA contains a restriction enzyme recognition sequence in the overhang of the first double-stranded DNA fragment, the adapter DNA may contain a base sequence complementary to the recognition sequence in the linking sequence. it can.

  The adapter DNA can further include an amplification sequence. The amplification sequence can determine the second double-stranded DNA fragment according to the mode of the amplification step in the subsequent amplification step. For example, in the case of PCR in which a so-called temperature cycle is performed, the primer can be a sequence for annealing. The length of the amplification sequence in this case is not particularly limited, but can be, for example, about 18 base lengths to 25 base lengths. The adapter DNA intended for PCR amplification may be a single-stranded DNA as shown in FIG. 1, but at the same time may have a double-stranded part as shown in FIG. By doing so, even when the overhang is short, it can be efficiently linked to the first double-stranded DNA fragment. When the adapter DNA has a double-stranded DNA portion, the double-stranded portion is complementary to the portion of the DNA strand adjacent to the linking sequence so that the linking sequence can be overhanged in the adapter DNA. Double-stranded DNA can be combined.

  In the adapter DNA of this embodiment, the variation of the base sequence of the amplification sequence may be one type or two or more types. The amplification sequence is preferably less diversified than the linking sequence. That is, it is preferable that the base sequence is more common than the linking sequence. By reducing the number of types of amplification sequences, the amplification efficiency can be made uniform and the amplification efficiency can be improved. By using a single type of amplification sequence, the amplification efficiency can be most uniformed and maximized. That is, the adapter DNA is preferably an adapter DNA capable of forming a nascent overhang having a base sequence common to the 3 'end and 5' end of the first double-stranded DNA fragment as an amplification sequence. In this way, only one type of primer can be used in the amplification process, and when the second double-stranded DNA fragment length is diversified over a wide range depending on the length of the first double-stranded DNA fragment Even so, the second double-stranded DNA fragment can be amplified uniformly and efficiently.

  In addition, when the isothermal amplification process using a strand displacement type DNA polymerase is performed in the subsequent amplification process, the adapter DNA is used in each strand of the second double-stranded DNA fragment, for example, as shown in FIG. A terminal hairpin structure can be formed at the end of the structure. That is, an adapter capable of providing a base sequence capable of forming a terminal hairpin structure on each of the 3 'end side and the 5' end side of the first double-stranded DNA fragment can be used. By forming such a terminal hairpin structure, it becomes possible to obtain an amplification product including a region other than the terminal repeatedly as shown in FIG. In addition to the linking sequence, such adapter DNA is complementary to a single-stranded DNA strand having a palindromic base sequence capable of forming a terminal hairpin structure as part of the amplification sequence, and the amplification sequence. In addition, a double-stranded part may be formed by providing a DNA strand that is paired so as to have the base sequence of the palindromic sequence as another part of the amplification sequence. In the adapter DNA of this embodiment as well, the linking sequence is formed in an overhang.

  Furthermore, the adapter DNA can include a base sequence suitable for transcription, transformation, and base sequence analysis. For example, a DNA region having a base sequence that can be used for sequencing, transcription reaction, transformation, intracellular or in vitro amplification, etc., which may be performed after the amplification step. For example, if a sequencing primer sequence is used, the product amplified in the subsequent amplification step can be directly sequenced. Such a base sequence can be appropriately included in the amplification sequence and the linking sequence in the adapter DNA, or can be added separately.

  From the above, one or two or more types of adapter DNA can be prepared depending on variations of the linking sequence and the amplification sequence.

  In this step, the adapter DNA and the first double-stranded DNA fragment are ligated to obtain a second Japanese-stranded DNA fragment. These ligations can be performed at a temperature (for example, about 10 ° C. or higher and 37 ° C. or lower) according to the type of enzyme using a DNA ligase such as T4 DNA ligase. As the DNA ligase, T4 DNA ligase or E. coli DNA ligase that operates at a relatively low temperature can be preferably used.

(Amplification process)
In the amplification step, the second double-stranded DNA fragment can be amplified using a DNA polymerase. Using the amplification sequence of the second double-stranded DNA fragment, the second double-stranded DNA fragment is amplified by a DNA polymerase, whereby the DNA to be amplified can be amplified. The mode of the amplification step using DNA polymerase can be appropriately determined depending on the mode of the adapter DNA amplification sequence used to obtain the second double-stranded DNA fragment. For example, when the second double-stranded DNA fragment has an amplification sequence derived from an adapter DNA that allows PCR primers to anneal, a known PCR can be performed using a primer complementary to the amplification sequence. Can be used to perform the amplification step. Primers are prepared according to the type of amplification sequence. When the amplification sequences at both ends of the second double-stranded DNA fragment have a single base sequence, By using a single kind of primer complementary to the amplification sequence, the second double-stranded DNA fragment can be efficiently and uniformly amplified. In particular, even when the length of the second double-stranded DNA fragment is abundant, uniform amplification is possible for each fragment. Those skilled in the art can appropriately set the conditions for such PCR process. The length of the amplification sequence in this case is not particularly limited, but can be, for example, about 18 base lengths to 25 base lengths.

  In addition, when DNA having an amplification sequence capable of forming a hairpin structure on each strand at both ends of the second double-stranded DNA fragment is used as the adapter DNA, isothermal amplification using a strand displacement type DNA polymerase A process (Liang XG et al. Biochemistry 43, 13459-13466, 2004) can be performed. By performing the isothermal amplification step, the amplification can be performed with simplified or omitted temperature control, so that the second double-stranded DNA fragment can be amplified uniformly. If adapter DNA having an amplification sequence capable of forming a hairpin structure is used, the end of the second double-stranded DNA fragment can have a hairpin structure. As the strand displacement type DNA polymerase, a known strand displacement type DNA polymerase such as Phi29 DNA polymerase can be used.

  According to this step, an amplification product of the second double-stranded DNA fragment can be obtained. As a result, the DNA to be amplified can be amplified.

  According to this amplification method, the first double-stranded DNA fragment obtained by cleaving the amplification target DNA using a restriction enzyme capable of forming an overhang of a specific aspect is prepared, and the overhang thus obtained is prepared. Utilizing the diversity of the base sequence. That is, by using the first double-stranded DNA fragment and the adapter DNA having the ligation sequence corresponding to the diversity of the overhang base sequence and the more common amplification sequence, They can be annealed and connected uniformly. As a result, a second double-stranded DNA fragment having an amplification sequence that is more common and suitable for amplification can be easily obtained. By amplifying the second double-stranded DNA fragment using the amplification sequence, the DNA to be amplified can be easily amplified with high efficiency. Therefore, according to this amplification method, a sufficient amount of DNA can be easily obtained from a small amount of DNA, such as DNA derived from one to several levels of cells.

  In particular, by increasing the commonality of amplification sequences, that is, by reducing the diversity of amplification sequences (most preferably by a single type), the types of adapter DNA and primers to be prepared are reduced. Thus, the acquisition process and the amplification process of the second double-stranded DNA fragment can be performed at a lower cost and higher efficiency.

  The amplification method is particularly effective when the amplification target DNA is long, such as a genome. For example, E. coli has genomic DNA with a total length of 4.6 million base pairs, but there are about 10,000 TspRI cleavage fragments, and the number of fragments having the same overhang is 20 or more. In contrast, in the case of a plasmid, only one fragment has the same overhang. That is, the DNA to be amplified becomes longer, the number of fragments cleaved by restriction enzymes increases, and the efficiency of amplification by ligation reaction and PCR increases. That is, as the number of cleaved fragments increases, ligation efficiency and PCR efficiency increase. In other words, the resulting double-stranded DNA fragment is amplified regardless of the sequence from the center, so that if the number of cleaved fragments increases and the concentration of double-stranded DNA fragments, i.e., ends, increases, the amplification efficiency improves. Become.

(DNA amplification kit)
The DNA amplification kit disclosed in the present specification includes one or two or more restriction enzymes which form an overhang having a base length of 5 or more bases and containing one or more arbitrary base sites, and the overhang One or two or more types of adapter DNAs having a linking sequence containing a base sequence complementary to the base sequence and an amplification sequence containing a common base sequence for amplification can be included. According to such a kit, the DNA amplification method disclosed in the present specification can be easily carried out.

  In the present amplification kit, the restriction enzyme, the first double-stranded DNA fragment, the adapter DNA, and the second double-stranded DNA fragment are all applied to the DNA amplification method disclosed in the present specification. Is done. Therefore, the mode of the adapter DNA varies depending on the type of restriction enzyme.

  When this amplification kit is intended for PCR amplification, the adapter DNA may be a single-stranded DNA as shown in FIG. 1, but at the same time has at least a double-stranded part as shown in FIG. It may be. By doing so, even when the overhang is short, it can be efficiently linked to the first double-stranded DNA fragment. When the adapter DNA has a double-stranded DNA portion, the double-stranded portion is complementary to the portion of the DNA strand adjacent to the linking sequence so that the linking sequence can be overhanged in the adapter DNA. It can be constructed by combining single-stranded DNA.

  When the amplification kit is intended for PCR amplification, a primer having a base sequence complementary to a common amplification sequence in the adapter DNA can be included. By doing so, an amplification kit suitable for the PCR process using primers can be obtained. The type of primer depends on the diversity of the amplification sequence contained in the adapter DNA. When two types of adapter DNAs are prepared for the amplification sequences, two types of primers corresponding to these two types of amplification sequences are prepared. When the adapter DNA has a single type of amplification sequence, a single type of primer is prepared. Furthermore, the amplification kit of this embodiment can also contain a DNA polymerase suitable for PCR.

  When the amplification kit is intended for isothermal amplification using a strand displacement type DNA polymerase, the adapter DNA can have a base sequence capable of forming a terminal hairpin structure as an amplification sequence. In the second double-stranded DNA fragment obtained using such adapter DNA, when a terminal hairpin structure is formed, synthesis of the nascent DNA strand by the strand-displacing DNA polymerase is started from the 3 'end. Furthermore, the amplification kit of this embodiment may contain a strand displacement type DNA polymerase.

  This amplification kit may contain a DNA ligase such as T4 DNA ligase for linking the first double-stranded DNA fragment and the adapter DNA.

(DNA detection method)
The method for detecting DNA disclosed in the present specification includes the first double-stranded DNA fragment preparation step, the second double-stranded DNA fragment acquisition step, and the amplification step described above, in addition to amplification. And an amplification product detection step in the step. According to this detection method, DNA to be amplified can be amplified with high efficiency, and the detection step can be performed using this amplification product. As a result, the detection step can be performed with high sensitivity. In the detection step, the detection target is not particularly limited, but one or more target DNAs contained in the amplification target DNA can also be detected.

  The detection method of the amplification product in the detection step of this detection method is not particularly limited. Examples of the general DNA detection method include electrophoresis, detection of amplification products using probes (including ordinary probes and other molecular beacon probes), and the like. For example, the probe may be bound to a solid phase such as an array. The probe may be accompanied by signal generation by a signal substance such as fluorescence. Moreover, such a detection process is not limited to performing only on the amplification product after the amplification process, and can be performed on the amplification product in the amplification process in real time.

  Hereinafter, the present invention will be described with specific examples, but the present invention is not limited to the following examples.

  In this example, plasmid pQBI T7-GFP (5115 bp) was amplified using the restriction enzyme TspRI as the amplification target DNA. Based on the disclosed nucleotide sequence of this plasmid, it is known that when cleaved with the restriction enzyme TspRI, 15 cleaved fragments are generated as shown in FIG. The lengths of the fragments are 1525, 63, 861, 360, 248, 113, 285, 242, 506, 13, 271, 149, 105, 347, and 27 base pairs, respectively, with an average of 341 bp.

The adapter and primer sequences used in this example were as follows. For each N in the adapter base sequence, an adapter was synthesized with a combination of all four bases.
Adapter: 5'-TCACACAGGAAACAGCTATGAC- NNCAC (G) TGNN-3 'Length 31 nt (SEQ ID NO: 1) Primer: 5'-TCACACAGGAAACAGCTATGAC-3' Length 22 nt (SEQ ID NO: 2)

The main reaction conditions in each step were as follows.
(1) First double-stranded DNA fragment preparation step Cleavage with restriction enzyme TspRI: 65 ° C., 3 h (50 mM sodium acetate, 20 mM Tris-acetic acid, 10 mM Mg (OAc) ₂ , pH 7.9 (25 ^° C), concentration of plasmid DNA 0.01pM -1 pM
(2) Second double-stranded DNA fragment acquisition step
Ligation with DNA T4 Ligase: 25 ° C, 16h (1 mM adapter DNA, 10 mM MgCl ₂ , 50 mM Tris-HCl, 1 mM ATP, 10 mM Dithiothreitol, pH 7.5 (25 ° C))
(3) Amplification process
PCR with Phusion DNA polymerase: 35 cycles (98 ° C for 10 seconds, 60 ° C for 30 seconds, 72 ° C for 45 seconds) (1 mM primer)

  Under the above conditions, amplification products (DNA) obtained by carrying out each step were analyzed by electrophoresis. The result is shown in FIG. As shown in FIG. 3 (b), DNA amplification could be confirmed even at a very low concentration of 0.1 pM. Furthermore, amplification up to 10 fM (0.01 pM) was confirmed.

  In this example, the entire genome of E. coli K-12 (4.64 million bp) was amplified using the restriction enzyme TspRI as the amplification target DNA. Based on the disclosed nucleotide sequence, it is known that there are 10344 cleavage sites when cleaved with the restriction enzyme TspRI, and the average is 450 bp per one cleavage site. Each step was performed in the same manner as in Example 1 except that this E. coli was used as it was as a sample. Since Escherichia coli is disrupted under the high temperature conditions of PCR, the disruption step was omitted. The results are shown in FIG. As shown in FIG. 4, it was confirmed that DNA obtained from several tens of E. coli can be amplified (see lane 1).

In this example, human genomic DNA (3 billion base pairs) was amplified using the restriction enzyme TspRI as the amplification target DNA. Each step was performed in the same manner as in Example 1 except that the supernatant of human 293FT cells (human cancerous kidney cells) was directly used as a sample. The results are shown in FIG. As shown in FIG. 5, it was confirmed that DNA could be amplified from about 100 human cells (lane 1), about 10 human cells (lane 2), and about 1 human cell (lane 3). According to this method, it has been found that the amplification efficiency increases when the DNA to be amplified becomes longer. That is, it was found that DNA can be amplified with sufficient efficiency even from one cell.
(Sequence table free text)
SEQ ID NOs: 1 and 2: Synthetic DNA

Claims (12)

A method for amplifying DNA comprising:
A first double-stranded DNA fragment is prepared by treating the DNA with one or more restriction enzymes having a base length of 5 bases or more and forming an overhang containing one or more arbitrary base sites. And a process of
Adapter DNA having a linking sequence containing a base sequence complementary to the overhang base sequence and an amplification sequence containing a common base sequence for amplification is ligated to the first double-stranded DNA fragment. And obtaining a second double-stranded DNA fragment;
Amplifying the second double-stranded DNA fragment using a DNA polymerase;
A method comprising:   The method according to claim 1, wherein the step of obtaining the first double-stranded DNA fragment comprises treating with the restriction enzyme that forms the overhang having a base length of 9 bases.   The method according to claim 2, wherein the restriction enzyme is TspRI.   The step of obtaining the second double-stranded DNA fragment can form a nascent overhang having the amplification sequence including a base sequence common to the 3 ′ end side and the 5 ′ end side of the double-stranded DNA fragment. The method according to any one of claims 1 to 3, which is a step of using the adapter DNA.   The method according to claim 4, wherein the amplification step is a step of amplifying the second DNA fragment by PCR using a primer complementary to the amplification sequence.   The step of obtaining the second double-stranded DNA fragment has the amplification sequence including a base sequence capable of forming a terminal hairpin structure on the 3 ′ end side and the 5 ′ end side of the double-stranded DNA fragment, respectively. The method according to any one of claims 1 to 3, which is a step of using the adapter DNA.   The amplification method according to claim 6, wherein the amplification step is a step of amplifying the second double-stranded DNA by isothermal amplification.   The preparation step of the first double-stranded DNA fragment is a step of preparing the first double-stranded DNA fragment by treating the DNA with one kind of the restriction enzymes. The amplification method according to claim 1. The step of preparing the first double-stranded DNA fragment is a step of preparing the second double-stranded DNA fragment by treating the DNA with two or more kinds of the restriction enzymes, respectively.
The step of obtaining the second double-stranded DNA fragment is a step of obtaining the second double-stranded DNA fragment for each of the first double-stranded DNA fragments obtained for two or more kinds of the restriction enzymes. And
The method according to any one of claims 1 to 7, wherein the amplification step is a step of performing an amplification step on the second double-stranded DNA fragment obtained for each of the two or more types of restriction enzymes. A kit for amplifying DNA,
One or more restriction enzymes having a base length of 5 or more bases and forming an overhang containing one or more arbitrary base sites;
An adapter DNA having a linking sequence comprising a base sequence complementary to the overhang base sequence and an amplification sequence comprising a common base sequence for amplification;
Including a kit.   The kit according to claim 10, further comprising a primer having a base sequence complementary to a common amplification sequence in the adapter DNA. The kit according to claim 10, wherein the adapter DNA has the amplification sequence including a base sequence capable of forming a terminal hairpin structure.