HK1035004A - Detection of sequence variation of nucleic acid by shifted termination analysis - Google Patents

Description

Detection of nucleic acid sequence variations by termination of movement analysis

The present invention relates to the field of nucleic acid sequence detection. The present invention relates to a method for detecting any type of mutation at a predetermined nucleic acid base site of interest. The methods referred to herein are referred to as mobile termination analysis, also known as specific termination analysis, or mobile terminator sequence alignment (which may be abbreviated as STA).

Practical applications of the methods of the invention include the diagnosis of genetic diseases, the diagnosis of infectious diseases, forensic techniques, paternity determinations (paternity determinations), and genome mapping, where the mutation sites to be detected are known.

Over the past decade, genes that are genetically predisposed to developing cancer have been identified, and many mutations associated with cancer have been identified. Diagnostic tests for these mutations provide a more accurate prediction of the likelihood of cancer in an individual. Early diagnosis of cancer-associated mutations is one of the objects of the present invention.

There are four major types of gene mutations. The first is a point mutation, which results from the substitution of a single nucleotide in the normal DNA sequence. In most cases, this mutation causes a reading frame shift in the coding strand, resulting in termination of normal protein synthesis. Point mutations in the APC gene found in patients with familial polyposis (FAP) are a typical example (Kinzler et al, Science 253, 661-. The second is insertion mutation in which a single or multiple nucleotides are inserted into a normal DNA sequence. The third is deletion mutation in which a single or multiple nucleotides are deleted in the normal DNA sequence. Both insertion and deletion mutations will cause serious changes, such as frame shifts, early termination of protein synthesis, and the addition or absence of one or more amino acids. The fourth is gene translocation, which occurs when a gene fragment is incorporated into another gene. The Philadelphia chromosome found in patients with chronic myelogenous leukemia is an example of this phenomenon (Konopka et al, Cell 371035 (1984)). The alteration of protein structure causes a series of disorders in the cells, leading to the onset of cancer.

It is difficult to detect a mutated DNA among countless normal DNAs. Chemical or enzymatic DNA sequencing methods, which directly read the sequence of an isolated DNA, have been used in research laboratories as the most accurate method for analyzing and identifying gene mutations. However, clinical application of this sequencing method is impractical because it suffers from the following limitations: the level of expertise, the amount of work required to perform such analyses, the high costs associated with purchasing instruments and reagents to complete the sequencing reactions, and the long periods required to complete the entire project. Finally, this sequencing method has another drawback: this procedure requires a large number of DNA templates, which are difficult to obtain from a 10ml blood sample taken from a patient.

Examples of conventional mutation detection methods include: restriction Fragment Length Polymorphism (RFLP) (Botstein et al, am. J. hum. Genet.,32,314-331(1980), and White et al, Scientific American, 258: 40-48 (1988)); single-stranded conformation polymorphism (SSCP) (Howell et al, am. J. hum. Genet.,55, 203-.

Some of these techniques are only suitable for detecting point mutations. Some of the remaining techniques can only be used to detect insertion or deletion mutations, such as disruption or construction of restriction enzyme cleavage sites, but are not suitable for detection of single base mutations. For example, point mutations that do not affect the cleavage site will not be detected using these techniques, such as RFLP. Other techniques require optimal conditions for specific probe hybridization. In addition, the above-mentioned techniques require special laboratory instruments such as gel electrophoresis equipment and hybridization equipment, time and labor.

Several methods based on primer extension are known for detecting mutations (Mohan et al, Proc. Natl. Acad. Sci. USA,88,1143-1147(1991), Prezant et al, hum. mutation 1,159-164(1992), Fahy et al, Nucleic Acid Research,25,3102-3109(1997), and U.S. Pat. Nos. 5,846,710(1998) and 5,888,819 (1998)). These methods include: primer extension with a thio nucleotide; primer extension from an oligonucleotide primer flanking the mutant nucleotide with a labeled nucleotide complementary to the mutant nucleotide base; and using a labeled dideoxynucleotide terminator complementary to the mutated base to perform primer extension.

These primer extension-based mutation detection techniques are rapid, easy to operate and potentially applicable to clinical applications. However, these methods have at least two drawbacks. First, all of these methods are based on the incorporation of only one labeled nucleotide into the primer extension strand. Incorporation of only one type of labeled nucleotide (selected from A, C, G, T or U), or labeled dideoxynucleotide, allows detection of only specific point mutations that are specific for the nucleotide base complementary to the labeled nucleotide used for the assay.

Given that when different types or properties of mutations occur at the same site, such as a to C or CT or TCT with many other changes, these known methods require at least three separate tests with labeled G, C, A. Alternatively, a single test assay may be used with different labeled nucleotides in conjunction with gel assays and specific label detection systems to detect G, C, A mutants separately. However, at least three blood samples are taken from the patient for three separate tests. Such multiple volumes of blood sample or complex gel analysis procedures not only add expense, time and labor to the test, but, more importantly, increase the chance of error due to the possibility of mislabeling of the tubes and numerous steps required to perform these analyses. Therefore, these primer extension-based methods are inconvenient or unsuitable for screening of large numbers of samples or for routine clinical experiments.

Second, the sensitivity of these primer extension-based assays needs to be improved. Since the primer extended strands obtained in these tests carry only one labeled nucleotide or labeled dideoxynucleotide, the signals generated are diverse and their intensity depends on the type of chemical label used. But in general, such signals are weak.

Therefore, there is a need in the art of mutation detection for a rapid, low cost, low workload and clinically applicable technique that can be used to detect any type of mutation that occurs at a specific site nucleobase and provide a strong and accurate detection signal.

The present invention meets the above-described need.

Although the present invention has some of the advantages of the conventional primer extension-based methods, such as simple design of the test for mutations at specific sites, the present invention provides methods that overcome the above-mentioned disadvantages associated with primer extension-based methods. The invention can be widely applied to the detection and identification of all types of mutations. It is cost effective, time saving and less labor intensive than conventional methods.

Some key advantages of the present invention over the above methods are 1) the ability to detect all types of mutations in one reaction tube without size separation by gel electrophoresis; 2) the strong signal resulting from the incorporation of multiple labeled nucleotides into the primer extension strand, resulting in high test sensitivity; and 3) high accuracy because two or three different nucleotides or nucleotide analogs can be labeled into the primer extension strand at the same time. These advantages make the present invention applicable to the routine test of any gene mutation existing in clinical due to its simple inventive procedure. The invention is also applicable to the automatic screening of large numbers of samples.

The present invention relates to a method for detecting any mutation occurring at a predetermined nucleotide (target base) in a known nucleic acid sequence in a reaction. The method of the invention uses primer extension analysis to detect mutations. Preferably, the primer is complementary to and sequence-specifically hybridizes to the nucleic acid at the site of interest adjacent to the preselected nucleotide base to form a duplex, such that the target base in the nucleic acid of interest becomes an unpaired base immediately downstream of the 3' end of the primer. The primer extension reaction reagents include one unlabeled terminator nucleotide type (or optionally no corresponding nucleotide base) and three labeled (or optionally differently labeled or unlabeled) non-terminator nucleotides, where the terminator nucleotide is complementary to the target base at the predetermined site of the nucleic acid of interest. The labeled non-terminator nucleotide is not complementary to the target base. Incorporation of a terminator nucleotide at the 3' end of the primer (which is complementary to the target base in the nucleic acid of interest) will terminate the primer extension reaction without further incorporation of any labeled non-terminator nucleotides. If the target base is altered due to mutation, the labeled non-terminator will be sequence-dependently incorporated into the primer. Thus, any marker signal detected by the primer will indicate that a mutation has occurred at the predetermined nucleobase site.

The object of the present invention is to provide a method for detecting or quantifying a target nucleic acid in a sample, comprising:

(a) preparing a primer complementary to a sequence immediately upstream of a target nucleotide base at a predetermined position in a nucleic acid template of interest;

(b) if the nucleic acid is double-stranded, processing the sample containing the nucleic acid of interest to obtain unpaired nucleotide bases spanning the specific position; directly using step (c) if the nucleic acid of interest is single stranded;

(c) annealing the target nucleic acid of (b) to the primer of (a) under high stringency conditions to provide a primer-nucleic acid duplex, wherein the target nucleotide base in the nucleic acid of interest is the first unpaired base immediately downstream of the 3' end of the primer;

(d) mixing a primer extension reaction reagent with the primer-nucleic acid duplex of (c), the primer extension reaction reagent comprising: (ii) a terminator nucleotide or optionally no nucleotide, which is complementary to the target base at the predetermined site in the nucleic acid of interest, and (ii) three non-terminator nucleotides different from the terminator nucleotide in (i), at least one of which is optionally labeled with a detectable label;

(e) enzymatically or chemically, wherein the terminator nucleotide or non-terminator nucleotide is incorporated into the primer, depending on the identity of the unpaired nucleotide base in the nucleic acid template immediately downstream of the 3' end of the primer, and incorporating in the sequence said terminator nucleotide which is complementary to said target nucleotide base in the nucleic acid of interest will terminate said primer extension without incorporating into the primer any labeled non-terminator nucleotide, wherein the primer is unlabeled, and further, when the target nucleotide base is changed to any other type of nucleotide, (ii) if mass spectrometry is used as the detection method, sequence-dependently incorporating into the primer via said primer extension reaction a non-terminator nucleotide complementary to the mutated nucleotide base, labeled with said detectable label or optionally without any label; and

(f) the presence and identity of the nucleotide base at the predetermined site in the nucleic acid of interest is determined by detecting the incorporation of the labeled non-terminator in the primer.

In a preferred embodiment, in step (b), the nucleic acid base of interest is adjacent to the nucleotide base to be identified at the predetermined site, and the nucleotide base to be identified is an unpaired base at the predetermined site immediately downstream of the 3' end of the duplex. In step (d) of a preferred embodiment, the duplex of step (c) is contacted with at least one labeled non-terminator and at least one unlabeled terminator. Further, in step (d), the duplex of step (c) is contacted with a non-terminator, wherein each non-terminator is labeled with the same or a different detectable label.

In another preferred embodiment, the above method can be practiced wherein the template-dependent enzyme is E.coli DNA polymerase I or Klenow fragment of E.coli DNA polymerase I, T4DNA polymerase, T7DNA polymerase, Thermus aquaticus DNA polymerase, retroviral reverse transcriptase, or a combination thereof.

In another preferred embodiment, the nucleic acid of the invention is deoxyribonucleic acid, ribonucleic acid or a copolymer of deoxyribonucleic acid and ribonucleic acid. The primer is an oligodeoxynucleotide, an oligoribonucleotide, or a copolymer of deoxyribonucleic acid and ribonucleic acid. The template is a deoxyribonucleic acid, the primer is an oligodeoxynucleotide, an oligoribonucleotide, or a copolymer of a deoxyribonucleotide and a ribonucleotide, and the template-dependent enzyme is a DNA polymerase. Preferably the template is a ribonucleic acid, the primer is an oligodeoxynucleotide, an oligoribonucleotide or a copolymer of a deoxyribonucleotide and a ribonucleotide, and the template-dependent enzyme is a reverse transcriptase. Preferably, the template is deoxyribonucleic acid, the primer is an oligonucleotide, and the enzyme is RNA polymerase. Preferably, the template is a ribonucleic acid, the primer is an oligoribonucleotide, and the template-dependent enzyme is an RNA replicase.

In step (d) of the above method, the duplex in step (c) is contacted with at least one labeled non-terminator and at least one terminator (labeled differently from the non-terminator). In a further step (e), the label signal of the incorporated labeled non-terminator and at least one terminator, which is labeled differently from the non-terminator, is detected.

According to the methods of the invention, the nucleic acid of interest can be synthesized enzymatically in vivo, synthesized enzymatically in vitro, or synthesized non-enzymatically. In another embodiment of the method of the invention, the oligonucleotide primer may be synthesized enzymatically in vivo, synthesized enzymatically in vitro, or synthesized non-enzymatically. In addition, the oligonucleotide primer may include one or more components that allow for affinity separation of the primer from unincorporated reagents and/or nucleic acids of interest.

In particular, in a preferred embodiment, the oligonucleotide primer comprises biotin, such that the primer can be affinity separated from the unincorporated reagent and/or the nucleic acid of interest by binding biotin to streptavidin, which is attached to a solid support. In another embodiment of the invention, the oligonucleotide primer sequence comprises a DNA sequence such that the primer can be affinity separated from the unincorporated reagent and/or the nucleic acid of interest by bases that pair with a complementary sequence present in the nucleic acid (attached to a solid support). In another embodiment of the invention, the nucleic acid of interest comprises one or more moieties that allow affinity separation of the nucleic acid of interest from unincorporated reagents and/or primers. The nucleic acid of interest can include biotin, which allows for affinity separation of the nucleic acid of interest from unincorporated reagents and/or primers by binding biotin to streptavidin, which is attached to a solid support.

In the methods of the invention, the sequence of the nucleic acid of interest comprises a DNA sequence such that the nucleic acid of interest can be affinity separated from the unincorporated reagent and/or primer by bases that pair with a complementary sequence in the nucleic acid (attached to a solid support). The oligonucleotide primers may be labeled with a detectable label. The oligonucleotide primer is labeled with a detectable label that is different from any detectable label in the reagent or attached to the nucleic acid of interest. The nucleic acid of interest can be labeled with a detectable label. The nucleic acid of interest is labeled with a preferred detectable label, which is different from the detectable label present in any of the reagents or attached to the primer.

In another embodiment of the invention, the nucleic acid of interest comprises a non-natural nucleotide analog. The non-natural nucleotide analogs include deoxyinosine or 7-deaza-2' -deoxyguanosine. The nucleic acid of interest can be synthesized by polymerase chain reaction.

In another method of the invention, the sample comprises genomic DNA from an organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof. The sample may comprise extra-genomic DNA of the organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof. In the method of the present invention, it is preferred that the primer is isolated from the nucleic acid of interest after the primer extension reaction of step (d) under suitable denaturing conditions. Preferred denaturing conditions include heat, alkali, formamide, urea, glyoxal, enzymes, and combinations thereof. More preferred denaturing conditions include treatment with 0.2N NaOH.

Nucleic acids for use in the practice of the methods of the invention are from any organism including: plants, microorganisms, viruses, or birds. The organism may be a vertebrate or an invertebrate. Preferably the organism is a mammal. More preferably the organism is a human. The mammal may also be a horse, dog, cow, cat, pig or sheep.

These and other objects of the present invention will be more fully understood from the following description of the invention, the accompanying drawings and the claims.

FIGS. 1A-1C are schematic diagrams showing a preferred embodiment of the mutation detection method of the present invention. "L" represents a wild-type nucleotide, including A, G, C, T, or U. "L" represents an unlabeled terminator, such as a dideoxynucleotide complementary to L. "M" represents a mutation at the L site, and the mutated nucleotides include A, G, C, T, or U. "W" represents a nucleotide complementary to M and may include A, G, C, T, or U, with a detectable label. "n" represents one or more nucleotides or nucleotide analogs, including A, G, C, T, and U. "y" represents a nucleotide or nucleotide analog, including A, G, C, T, or U, labeled with a detectable label and complementary to M or n.

Fig. 2.

Mu.l of the STA reaction mixture was spotted on a thin layer chromatography plate, and the plate was then placed in a solvent containing 1M NaCl and 1M HCl. The chromatographic strips were dried at room temperature for 10 minutes and exposed to Kodak film for 30 minutes, which was developed by an automatic film developer. The templates used for the STA test were marked under each slice. The upper arrow shows the free nucleotide front and the lower arrow shows the incorporation-³²P]Primer extension of dCTP.

As used herein, a "nucleic acid" or "nucleotide" can be a deoxyribonucleic acid, a ribonucleic acid, or a copolymer of deoxyribonucleic acid and ribonucleic acid. The nucleic acid sample may be natural or synthetic. The nucleic acid sample may be a nucleic acid of natural origin or may be derived from any organism. Some organisms that can be used in the methods of the invention are: plant, microorganism, virus, bird, vertebrate, invertebrate, mammal, human, horse, dog, cow, cat, pig or sheep. The target nucleic acid can be natural, or can be in vivo enzymatic synthesis, in vitro enzymatic synthesis or non-enzymatic synthesis.

The sample containing a nucleic acid or nucleic acids of interest may contain genomic DNA from an organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof. A sample containing a nucleic acid or nucleic acids of interest may also contain DNA outside the genome of the organism, their RNA transcripts, or cDNA prepared from these RNA transcripts. Likewise, the nucleic acid of interest or the nucleic acids of interest may be synthesized by polymerase chain reaction.

The nucleic acid of interest may include non-natural nucleotide analogs such as deoxyinosine or 7-deaza-2' -deoxyguanosine. These analogs destabilize the DNA duplex and allow annealing to the primer, allowing extension reactions to occur in double-stranded samples without complete strand separation.

The nucleic acid of interest may include one or more components that allow for affinity separation of the nucleic acid of interest from unincorporated reagents and/or primers. For example, the nucleic acid of interest can contain biotin, such that the nucleic acid of interest can be affinity separated from the unincorporated reagent and/or primer by binding biotin to avidin or an analog thereof (attached to a solid support). The nucleic acid sequence of interest may comprise a DNA sequence that can be affinity separated from unincorporated reagents and/or primers by bases that pair with complementary sequences present in the nucleic acid (attached to a solid support). The nucleic acid of interest can be labeled with a detectable label; the detectable label is different from any detectable label present in the reagent or attached to the primer.

The "normal nucleotide" or "normal base" herein is a wild-type or previously known standard nucleotide base identified at the base site from which a mutation is examined. "Standard nucleotide base" includes any known base which may be a wild-type or known mutant base (so long as the base is known and it is desired to know variants thereof). Thus, for example, the normal base may be a known wild-type base, and a mutation is sought at that site. In contrast, the known base may be a known mutant, and the presence of the wild type base is sought at that site. Alternatively, the known normal base may be a known mutant for which another mutant variant base is sought. Thus, the method of the invention can be applied to any known sequence for determining the presence of any other base variant at that site.

The term "primer" or "oligonucleotide primer" herein refers to an oligonucleotide that has the ability to serve as a point of initiation of synthesis when subjected to conditions that allow synthesis of a primer extension product complementary to a nucleic acid (template) strand in the presence of various factors such as nucleotides and enzymes (e.g., DNA polymerase) and suitable temperature and pH.

The term "primer" can also be defined as any nucleic acid fragment obtained from any source. For example, primers can be generated by fragmenting larger nucleic acid fragments (e.g., genomic DNA, cDNA, or DNA from PCR). That is, the nature of the primer is not limited by how the primer is obtained, whether it is a fragmented natural or synthetic nucleic acid or from a synthetic nucleic acid primer. In addition, the primer may be an oligodeoxyribonucleotide, an oligodeoxyribonucleotide copolymer, an oligoribonucleotide, a ribonucleotide copolymer, or a deoxyribonucleotide and ribonucleotide copolymer. The primer may be natural or synthetic. Oligonucleotide primers can be synthesized enzymatically in vivo, synthesized enzymatically in vitro, or synthesized non-enzymatically in vitro. The primer may be labeled with a detectable label; the detectable label is different from any detectable label in the reagent or attached to the nucleic acid of interest. In addition, the sequence of the primer must correspond to the sequence flanking the particular location of interest, either adjacent to or upstream of the nucleotide base being identified.

In addition, the primer must have the ability to hybridize or anneal to a nucleotide in the nucleic acid of interest. One way to achieve the desired hybridization is to have the template-dependent primer be sufficiently or completely complementary to a known base sequence.

The oligonucleotide primer may include one or more moieties that allow for affinity separation of the primer from unincorporated reagents and/or nucleic acids of interest. These affinity components include, but are not limited to, digitonin, magnetic beads, and ligands, such as protein ligands (including antibodies). The preferred composition is biotin. In experiments with biotin, primers containing biotin can be affinity separated from unincorporated reagents and/or nucleic acids of interest by binding biotin to avidin and its analogs (attached to a solid support). The oligonucleotide primer sequence may comprise a DNA sequence that permits affinity separation of the primer from the unincorporated reagent and/or the nucleic acid of interest by base pairing with a complementary sequence in the nucleic acid attached to a solid support.

The term "primer extension reaction" as used herein refers to a reaction in which a template-dependent nucleic acid synthesis reaction can be carried out under reaction conditions. Conditions under which the template-dependent primer extension reaction occurs can be created, in part, by the presence of an appropriate template-dependent enzyme. Some suitable template-dependent enzymes are DNA polymerases. DNA polymerases can be of many types. However, the DNA polymerase must be primer and template dependent. For example, E.coli DNA polymerase I or the Klenow fragment of E.coli DNA polymerase I, T4DNA polymerase, T7DNA polymerase ("sequencer enzyme"), Thermus aquaticus DNA polymerase, or retroviral reverse transcriptase may be used. Also in some procedures, RNA polymerases such as T3 or T7 RNA polymerase may be used. Different polymerases must use different conditions and different temperature ranges in hybridization and extension reactions.

The term "primer-extended strand" as used herein includes a strand that forms opposite to the template in a double strand after a primer is added. Preferably, primer extension is terminated by binding of a terminator to the template.

As used herein, the term "template" refers to a nucleic acid, including double-stranded DNA, single-stranded DNA, and RNA, or any modification thereof, and may be of any length or sequence.

As used herein, the term "terminator" or "chain terminator" refers to a nucleobase, such as A, G, C, T, or U, or an analog which is effective in terminating a primer extension reaction when incorporated into a primer extension strand opposite a template strand. Preferred terminators are dideoxynucleotides. Also preferably, the terminator is either unlabeled or labeled, but is distinguished from labels other than terminators. When the term "terminator" or "chain terminator" is used herein in the singular, it does not mean that a single nucleotide molecule is used. The singular form of the term "terminator" refers to the type of nucleotide, nucleic acid base, or nucleic acid analog being analyzed. For example, if the terminator is ddA, the singular form refers to all ddA as a whole, not a single molecule of ddA. In addition, a "terminator" can be the absence of a particular type of nucleotide, such that primer extension can be terminated by the absence of a particular nucleotide at a locus. For example, if it is desired that the primer extension reaction terminate opposite a "C" on the template strand, the non-terminating bases A, T and C should be included in the primer extension reaction mixture, but not the "G" (complementary to "C"). Therefore, the absence of complementary base will terminate the primer extension reaction with the same effect as adding dideoxy terminator nucleotides.

As used herein, the term "non-terminator" or "non-chain terminator" includes nucleotide bases that do not terminate a primer extension reaction when incorporated into a primer extension strand. Preferably, at least one non-terminator is labeled in the primer extension reaction. As used herein, when the term "non-terminator" or "non-chain terminator" is used in the singular, it does not mean that a single nucleotide molecule is used. The singular form of the term "non-terminator" refers to the type of nucleotide, nucleic acid base, or nucleic acid analog being analyzed. For example, if the terminator is G, the singular form refers to the entirety of all G, not to a single molecule G.

The term "mutant" or "mutation" as used herein refers to any base on the template strand that is different from the wild-type or normal base. The mutation detected by the method of the invention may be any type of mutation, including a single base mutation, insertion, deletion, or gene translocation, provided that the base on the template opposite the base immediately 3' to the annealed primer is affected.

The term "label" as used herein refers to any molecule attached to a terminator or non-terminator to provide a detectable signal. The label can be radioactive, chemiluminescent, a protein ligand such as an antibody, or if a fluorophore is used, a different fluorophore can be used for each type of non-terminator nucleotide base. The emission spectra of these fluorescent markers should be distinguishable.

In addition, methods for determining the level of incorporation of nucleotide bases in primer extension products can be determined by mass spectrometry techniques, as exemplified in U.S. Pat. No.5,885,775, which is incorporated herein by reference.

As used herein, the phrase "highly stringent hybridization conditions" refers to nucleic acid hybridization conditions such as, but not limited to, washing conditions with 0.1XSSC at 42 ℃. Hybridization conditions are generally found in conventional Molecular Biology manuals, such as Current Protocols in Molecular Biology, Greene and Wiley, pub. (1994), by Ausubel et al, which is incorporated herein by reference.

As used herein, "Thin Layer Chromatography (TLC)" may be performed on a paper medium based on a cellulose product, but may be comprised of any substance that allows the molecules to disperse well and form a uniform layer. Such materials include, but are not limited to, inorganic materials such as silica gel, alumina, diatomaceous earth, or magnesium silicate. Organic materials include, but are not limited to, cellulose, polyamide or polyethylene powders. Thin layer chromatography methods are generally described in handbooks of chemistry, such as set forth in Freifelder, Physical Biochemistry-application Biochemistry and Molecular Biology, second ed., published by Freeman and Co (1982), incorporated herein by reference, and in particular Chapter 8 at page 229-.

One skilled in the art will appreciate that, unlike non-terminator labeled terminators, it can be used to distinguish between incorporation of a terminator or a non-terminator in a primer extension strand. For simplicity of explanation in this application, the terminator is exemplified as lacking a particular type of nucleotide, but the description is not intended to limit the claims in any way. The invention also includes differently labeled or unlabeled terminators provided that the label on the terminator is different from the label on the non-terminator.

It will also be understood by those skilled in the art that a primer can be designed that binds to the template strand such that binding of the primer to the template strand occurs, provided that the sequence of the template is at least partially known. It will also be appreciated by those skilled in the art that the method of the invention may be practiced by using several primers in one or more assay tubes.

One feature of the method of the invention is that strong signals can be generated if the non-terminators are labeled the same, since incorporation of several labeled non-terminators into the primer extension strand will generate signals that add when there is a variation at the predetermined site. Which produces more favorable signal intensity than conventional variation detection methods (incorporating only one signal label per primer extension strand). When different labels are specific for each terminator or non-terminator, the signal is observed to improve accuracy.

The following examples are intended to illustrate the invention without limiting the claims in any way.

Examples

Example 1

The human APC gene sequence was selected as the target sequence for the STA test in the invention. Oligonucleotides corresponding to the wild-type APC sequence 4317-4347 and three different types of mutations were synthesized and used as templates. The primers used in the STA test are listed in Table 1.

TABLE 1

Name (R)	Sequence of	Description of the invention
			Form panel
Oapc-w	5′CCTGGACAACCATGCCACCAAGCAGAAGTA(SEQIDNO：1)	Wild type
			Oapc-p	5′CCTGGAtAACCATGCCACCAAGCAGAAGTA(SEQID NO：2)	Point mutation
Oapc-i	5′CCTGGAtgtAACCATGCCACCAAGCAGAAGTA(SEQIDNO：3)	Insertion mutation
			Oapc-d	5′CCTGG……AACCATGCCACCAAGCAGAAGTA(SEQIDNO：4)	Deletion mutations

STA primer STA0902

TTGGTACGGTGGTTCGTCTT 5’(SEQIDNO：5)

STA: each STA reaction was performed in 20. mu.l buffer (10mM Tris-HCl, pH7.5,50mM KCl, and 5mM MgCl)₂) The buffer solution contains 50ng of template oligonucleotide, 1. mu.M of primer, 2 units of DNA polymerase, and 1. mu.l of [ alpha-³²P]Labeled dCTP (20. mu. Ci/ml,3000Ci/mmol Dupont-NewEngland Nuclear), dATP, dTTP and 1. mu.l of unlabeled dd GTP. The mixture was incubated at 37 ℃ for 30 minutes and then heated at 100 ℃ for 3 minutes. Mu.l of the reaction mixture was applied to a TI strip, which was used for thin layer chromatography (TRIM USA, MD). The pieces were extended with a solution containing 1M HCl and 1M NaCl for 10 minutes. This procedure was used to completely separate the primers from unincorporated nucleotides on the TI strip. The labeled primers were visualized by autoradiography and radioactivity was counted using a scintillation counter (Beckman LS 5000). Fig. 2 shows an autoradiogram, and the counting results corresponding to the autoradiogram are shown in table 2.

TABLE 2

Form panel	Total count	Labeled primers
			Without form	76,675	95
Oapc-w	79,599	117
			Oapc-p	82,584	4,821
Oapc-i	75,376	8,602
			Oapc-d	100，634	6，571

The counts shown in Table 2, right column for incorporation into primer extension [. alpha. -³²P]The amount of dCTP, a wild-type oligonucleotide Oapc-w, was used as template. The first unpaired nucleotide after primer annealing is C, which is complementary paired to the terminator ddG. When the template-dependent primer extension reaction begins, the terminator ddG is rapidly incorporated at the 3' end of the primer as the first extended nucleotide, blocking further incorporation of the labeled nucleotide by the bound ddG. As a result, the primer is extended by only one nucleotide base, which is the terminator nucleotide. Since other nucleotide bases are unlikely to bind to the primer after the terminator binds, the primer extension reaction is terminated. The radioactive count of the Oapc-w sample showed that the count was similar to the sample without any template, i.e., the background control.

In contrast, Oapc-p is a point mutation oligonucleotide. The template is made by replacing the wild type C of the first unpaired nucleotide at the 3' end of the wild type template with the mutant T. In this experiment, dATP replaces the terminator ddG and at the beginning of the primer extension reaction, complementarily pairs with the mutated nucleotide T. After the terminator ddG is incorporated into the position opposite to the C residue of the template strand, the primer extension reaction is terminated.

In oligonucleotides comprising insertion mutationsIn the acid mutant, Oapc-i of tables 1 and 2, the first unpaired nucleotide is T, which is not complementary to the terminator ddG. In this experiment, the primer was extended by binding to the primer extension strand by the opposite dATP, and then the primer was further extended by adding: [ alpha-³²P]dCTP, dATP, two [ alpha ]^-32P]dCTP and dATP. Sequential incorporation of ddG by nucleotide polymerase terminates the extension process when it first encounters a C. The final result is three [ alpha-³²P]dCTP is incorporated into the primer extension strand.

Like the insertion mutation, the oligonucleotide deletion mutation, Oapc-d (tables 1 and 2), can be analyzed using the STA reagent and method of the invention. Extension of the primer through two [ alpha-³²P]dCTP and dATP, terminated with ddG. Thus, two [ alpha-³²P]dCTP is incorporated into the primer extension strand. These results provide strong evidence that the STA method of the invention can detect all types of mutations. The present STA method will identify the presence of any type of mutation by performing only one test.

In particular, in deletion and insertion mutations (Oapc-I and Oapc-d), multiple labeled nucleotides are incorporated into the primer extension strand. Such multiple labels significantly improve the detection sensitivity. In addition, the sensitivity of the assay can be further increased by using different nucleotides (labeled with the same detectable label). For example, all non-terminators in the extension primer may be labeled, e.g. [ alpha-³²P]-CTP、[α-³²P]-ATP、[α-³²P]-TTP。

Multiple labeling also offers the possibility of labeling non-terminator nucleotides with different detectable labels to distinguish each non-terminator nucleotide base. For example, the nucleotides may be labeled with different fluorescent dyes, and then the primers extended to carry different fluorescent labels. The detection of different signals at the same time will increase the accuracy of the STA test. These advanced multiple labeling features associated with the STA reagents and methods of the invention provide better sensitivity and accuracy of mutation detection than methods known in the art.

Example 2

The PCR product of the human APC gene was used as a test sample. PCR amplification of APC gene fragments was performed using standard PCR protocols. Human APC cDNA was used as template. The primers used for PCR are listed in Table 3.

TABLE 3

Primer and method for producing the same	Direction	Description of the invention
			5’TCCACCTGAACACTATGTTC(SEQIDNO：6)	Forward direction	Wild type
5’AGGTGGTGGAGGTGTTTTACTTCTGCTTGGCGGCA(SEQIDNO：7)	Reverse direction	Wild type
			5’AGGTGGTGGAGGTGTTTTACTTCaGCTTGGCGGCA(SEQIDNO：8)	Reverse direction	Mutation of T point to A
5’AGGTGGTGGAGGTGTTTTACTTCgcaGCTTGGCGGCA(SEQIDNO：9)	Reverse direction	Insertion mutation GCA

5’AGGTGGTGGAGGTGTTTTACTTC……GGCGGCATGGT(SEQIDNO：10)

Reverse direction

Deletion mutant TGCTT

Four different PCR products of about 200bp were generated by combining the primers. They are APC-w: a wild type; APC-p: point mutation; APC-i: insertion mutation; APC-d deletion mutation. The PCR product was loaded on a 1% agarose gel to remove the template and free nucleotides. The product was then purified using the Qiax DNA purification kit (Qiagen). The STA primer was designed to be 5'-AGGTGGTGGAGGTGTTTTACTTC-3' (SEQ ID NO: 11), and the STA reaction was performed in a total volume of 20. mu.l of buffer containing 10mM Tris-HCl, pH 8.3,50mM KCl,2mM MgCl₂0.05pmol of double-stranded PCR product, 5pmol of primer, 20pMdATP, dGTP, 1. mu. Ci [. alpha. -³²P]Labeled CTP,20 μ M unlabeled dideoxy TTP and 2 units of Taq DNA polymerase. 20 cycles (94 ℃,20 seconds; 55 ℃,1 minute) were carried out in a thermal cycler (Perkin Elmer, GeneAmp 9600). Mu.l of the STA product was loaded onto a Trim plate (thin layer chromatography plate manufactured by TRIM Corporation, Japan) and subjected to radioactive counting as described in example 1. The results are shown in Table 4.

TABLE 4

	Total count	Labeled primers
			Without form	182,245	343
Papc-w	208,271	595
			Papc-p	197,494	5,568
Papc-i	176,984	10,372
			Pape-d	195,570	12.010

By [ alpha ]³²P]dCTP extended the primers in all three types of mutant samples. The intensity of the signal generated by extension of the primer incorporating the label is well correlated with the number of labeled nucleotides (carried on the primer extension strand).

Example 3

The STA reagent and the method of the invention are applied to RNA fragments of human APC gene. The PCR product of the human APC gene of example 2 was ligated into TA cloning vector 3.1(TA cloning kit, Invitrogen). 4 vectors were constructed and are listed in Table 5.

TABLE 5

Carrier	Insert into	Description of the invention	Name of RNA product
				Tapc-w	APC-w	Wild type	Rapc-w
Tapc-p	APC-p	Point mutation	Rapc-p

Tapc-i	APC-i	Insertion mutation	Rapc-I
				Tapc-d	APC-d	Deletion mutations	Rapc-d

RNA corresponding to each vector was synthesized using an in vitro RNA synthesis kit (Promega, Wis.). RNA was synthesized at 37 ℃ for 1 hour in a buffer containing 2. mu.g of vector and T7 polymerase. The reaction was stopped by adding LiCl and 100% ethanol. After incubation at-20 ℃ for 15 minutes, the RNA was precipitated by centrifugation (14,000g,15 minutes) and the purified RNA was suspended in RNase-free water. Mu.g of gRNA were mixed with the STA primer described in example 2 in a total volume of 10. mu.l of buffer containing 10mM Tris-HCl pH7.6,50mM NaCl and 10mM KCl. The mixture was denatured by heating at 65 ℃ for 3 minutes and then quenched in ice for 2 minutes. STA reaction was performed as described in example 1, with 1. mu.l of [ alpha ] -containing buffer³²P]Labeled dCTP (250. mu. Ci/ml,3000Ci/mmol Dupont-New England Nuclear), 10. mu.M dATP, dGTP and 10. mu.M unlabeled ddTTP, and 20 units of reverse transcriptase. After incubation at 40 ℃ for 15 minutes, the reaction was terminated by heating at 100 ℃ for 2 minutes. Mu.l of the reaction product was loaded onto a Trim plate and radioactivity was counted as described in example 1. The results are shown in Table 6.

TABLE 6

Sample (I)	Total number of	Labeled primers
			Without form	167,496	690
Rapc-w	172,734	435
			Rapc-p	166,979	1,745
Rapc-i	170,801	7,348
			Rapc-d	174,888	7,360

All of the steps described above, including chemistry, operations and procedures, are or can be automated. Thus, incorporating the preferred mode of the invention into the operation of a suitably programmed robotic work cell would provide significant cost savings and would increase the efficiency of any diagnostic procedure (which relies on detecting specific nucleotide sequences or sequence differences in nucleic acids obtained from biological samples).

All references cited herein are incorporated by reference in their entirety.

Claims (36)

1. A method for detecting or quantifying a target nucleic acid in a sample, comprising:

(a) preparing a primer complementary to a sequence immediately upstream of a target nucleotide base at a predetermined position in a nucleic acid template of interest;

(b) if the nucleic acid is double-stranded, processing the sample containing the nucleic acid of interest to obtain unpaired nucleotide bases spanning the specific position; directly using step (c) if the nucleic acid of interest is single stranded;

(c) annealing the target nucleic acid of (b) to the primer of (a) under high stringency conditions to provide a primer-nucleic acid duplex, wherein the target nucleotide base in the nucleic acid of interest is the first unpaired base immediately downstream of the 3' end of the primer;

(d) mixing a primer extension reaction reagent with the primer-nucleic acid duplex in (c), the primer extension reaction reagent comprising: (ii) a terminator nucleotide or optionally no nucleotide, which is complementary to the target base at the predetermined site in the nucleic acid of interest, and (ii) three non-terminator nucleotides different from the terminator nucleotide in (i), at least one of which is optionally labeled with a detectable label;

(e) enzymatically or chemically, wherein the terminator nucleotide or non-terminator nucleotide is incorporated into the primer, depending on the identity of the unpaired nucleotide base in the nucleic acid template immediately downstream of the 3' end of the primer, and incorporating in the sequence said terminator nucleotide which is complementary to said target nucleotide base in the nucleic acid of interest will terminate said primer extension without incorporating into the primer any labeled non-terminator nucleotide, wherein the primer is unlabeled, and further, when the target nucleotide base is changed to any other type of nucleotide, (ii) if mass spectrometry is used as the detection method, sequence-dependently incorporating into the primer via said primer extension reaction a non-terminator nucleotide complementary to the mutated nucleotide base, labeled with said detectable label or optionally without any label; and

(f) the presence and identity of the nucleotide base at the predetermined site in the nucleic acid of interest is determined by detecting the incorporation of the labeled non-terminator in the primer.

2. The method of claim 1, wherein the primer is a fragment of deoxyribonucleic or ribonucleic acid, an oligodeoxyribonucleotide, an oligoribonucleotide, or a copolymer of deoxyribonucleic acid and ribonucleic acid.

3. The method of claim 1, wherein the nucleic acid of interest is deoxyribonucleic acid, ribonucleic acid, or a copolymer of deoxyribonucleic acid and ribonucleic acid.

4. The method of claim 1, wherein the target nucleotide is defined as any known base, including wild-type or known mutant bases, provided that the base is known and it is desired to know its variation.

5. The method of claim 1, wherein the terminator nucleotide is a dideoxyribonucleotide and the non-terminator nucleotide is a deoxyribonucleotide or a ribonucleotide.

6. The method of claim 1, wherein the terminator nucleotide is unlabeled.

7. The method of claim 1, wherein the terminator nucleotide is labeled with a detectable label that is different from the label in the non-terminator.

8. The method of claim 1, wherein in step (d) the duplex of step (c) is contacted with non-terminator nucleotides, and each non-terminator is labeled with the same or a different detectable label.

9. The method of claim 1, wherein the detectable label is an enzyme, a radioisotope, a fluorescent molecule, or a protein ligand.

10. The method of claim 1, wherein said detecting is by mass spectrometry.

11. The method of claim 1, wherein the enzyme is template-dependent.

12. The method of claim 11, wherein the template-dependent enzyme is a DNA polymerase.

13. The method of claim 12, wherein the DNA polymerase is e.coli DNA polymerase I or Klenow fragment of e.coli DNA polymerase I, T4DNA polymerase, T7DNA polymerase, or thermus aquaticus DNA polymerase.

14. The method of claim 11, wherein the enzyme is an RNA polymerase or a reverse transcriptase.

15. The method of claim 1, wherein the primer comprises one or more moieties that permit affinity separation of the primer from unincorporated reagents and/or nucleic acids of interest.

16. The method of claim 1, wherein the primer comprises one or more moieties that allow the primer to be attached to a solid surface.

17. The method of claim 15, wherein said composition comprises biotin or digitonin.

18. The method of claim 16, wherein said composition comprises biotin and digitonin.

19. The method of claim 15, wherein the moieties comprise DNA or RNA sequences such that the primer can be affinity separated from the unincorporated reagent and/or the nucleic acid of interest by base pairing to a complementary sequence in the nucleic acid attached to the solid support.

20. The method of claim 16, wherein the moieties comprise DNA or RNA sequences that allow affinity separation of the primer from the unincorporated reagent and/or the nucleic acid of interest by base pairing to a complementary sequence in the nucleic acid attached to the solid support.

21. The method of claim 15, wherein the moiety comprises a DNA or RNA sequence that allows the primer to be attached to the solid support by base pairing to a complementary sequence present on the solid surface.

22. The method of claim 16, wherein the moiety comprises a DNA or RNA sequence that allows the primer to be attached to the solid support by base pairing to a complementary sequence present on the solid surface.

23. The method of claim 1, wherein the nucleic acid of interest is synthesized enzymatically in vivo, in vitro, or non-enzymatically.

24. The method of claim 1, wherein the nucleic acid of interest is synthesized by polymerase chain reaction.

25. The method of claim 1, wherein the nucleic acid of interest comprises a non-natural nucleotide analog.

26. The method of claim 25, wherein the non-natural nucleotide analog comprises deoxyinosine or 7-deaza-2' -deoxyguanosine.

27. The method of claim 1, wherein the sample comprises genomic DNA of an organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof.

28. The method of claim 1, wherein the sample comprises extragenomic DNA of the organism, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof.

29. The method of claim 27, wherein the organism comprises a plant, a microorganism, a bacterium, a virus.

30. The method of claim 28, wherein the organism comprises a plant, a microorganism, a bacterium, a virus.

31. The method of claim 27, wherein the organism comprises a vertebrate or an invertebrate.

32. The method of claim 28, wherein the organism comprises a vertebrate or an invertebrate.

33. The method of claim 27, wherein the organism is a mammal.

34. The method of claim 28, wherein the organism is a mammal.

35. The method of claim 27, wherein the organism is a human.

36. The method of claim 27, wherein the organism is a human.