JP2023000571A - Method for purifying terminal-modified target nucleic acid - Google Patents

Abstract

To improve the sensitivity of genetic analysis or improve detection efficiency for genetic mutations, by selectively purifying primers having modified molecules at their ends.SOLUTION: Provided is a method for purifying a target nucleic acid having a modified molecule at its end, in a method for detecting a target nucleic acid or determining a target nucleic acid based on the mobility of the nucleic acid in electrophoresis, the method comprising: subjecting a double-stranded nucleic acid having a modified molecule at the end of one of the nucleic acid strands to a heat denaturation treatment to form a single-stranded nucleic acid, and treating the resulting single-stranded nucleic acid with an exonuclease at a heat denaturation temperature to remove nucleic acids other than the nucleic acid strand having the modified molecule at the end.SELECTED DRAWING: Figure 5

Description

The present invention relates to methods and kits for purifying target nucleic acids having modified molecules at their ends. The present invention also relates to a method for detecting a target nucleic acid or determining the base of a target nucleic acid using such a purification method.

With the progress of cancer research, the importance of detecting tumor-derived gene mutations by gene analysis technology is increasing in the field of cancer testing. In particular, a test that detects tumor-derived gene mutations in blood for medical diagnosis is called liquid biopsy, and it is expected to respond to applications such as early diagnosis of cancer, optimization of post-operative treatment options, and monitoring of residual tumors. Expected.

Tumor-derived genetic mutations released into the blood are known to have a low concentration and low mutation ratio, and high-sensitivity detection methods are required to detect low-concentration and low-frequency mutations. It is Therefore, prior to detection of gene mutations by each detection device, a pretreatment method called amplicon method or capture method is used to selectively concentrate target gene mutations or regions. The selectively enriched sample is detected by various detection means as a next step.

For example, in the next-generation sequencing method, which is currently most widely used as a means for detecting gene mutations, high-sensitivity detection of many types of gene mutations is a representative method. On the other hand, the test cost of the next-generation sequencer method is higher than that of other methods, and cost constraints are an issue in the spread of liquid biopsy. Therefore, for diagnostic applications in which the number of gene mutations to be detected is narrowed down, a low-cost test method has been developed using the PCR method, which has low test costs. Such diagnostic applications include, for example, companion diagnostics for judging drug indications in the treatment of lung cancer and colorectal cancer. has already been approved by regulatory authorities around the world.

In recent years, along with the progress of clinical tumor research, the number of biomarker items to be tested has expanded from dozens to thousands with the discovery of new ways to use tumor-related gene mutations that can be used as biomarkers. be. Especially in cancer early diagnosis, there is a trend toward early diagnosis by measuring many kinds of gene mutations. For example, in Non-Patent Document 1, the next-generation sequencer method is used to detect 1933 mutation sites in 16 genes, and the combination with the conventional tumor marker method leads to early cancer diagnosis. . In this way, the number of gene mutations to be targeted far exceeds the number of mutations that can be covered by the PCR method, and a detection method that can detect many types of gene mutations at low cost is desired. It is rare.

U.S. Patent No. 5,888,819

J. D. Cohen, et al., Science, 359, 926-930 (2018). D. Dias-Santagata, et al., EMBO Molecular Medicine, 2, 146-158 (2010).

Fragment analysis using capillary electrophoresis (CE) is an example of low-cost technology that can detect a wide variety of gene mutations (Fig. 1). This technique uses a selective primer 100 designed to change the electrophoretic distance so that each target mutation can be distinguished, and designed to be complementary to the gene sequence 101 containing the mutation to be detected. Using such a primer, if a target gene mutation exists, a polymerase synthesis reaction is performed at the 3′ end position of the primer corresponding to the gene mutation (typically a single nucleotide polymorphism, SNP). Dideoxynucleotides (ddNTPs) modified with four fluorescent dyes 102 are provided. Such a technique is called a single-base extension reaction (Patent Document 1). By single-base extension reaction, the extended primer for each gene mutation with a modified molecule at the end is converted to single-stranded DNA, and then separated by electrophoresis using capillary electrophoresis, and the fluorescent dye at the 3' end is detected by fluorescence. By doing so, gene mutations are finally detected.

For example, a method for detecting a total of 58 tumor-derived gene mutations using this technique has been reported (Non-Patent Document 2). However, in this document, the number of mutations that can be detected simultaneously by one reaction assay and electrophoresis is 5 to 8, and a total of 58 genes are detected by performing this analysis 8 times. Therefore, it is practically only possible to simultaneously detect gene mutations of several types. This is because in the conventional method, the upper limit of electrophoretic separation of the selective primer is limited to about 120 bases, and the usable electrophoretic region is small. Therefore, the conventional fragment analysis method using capillary electrophoresis has a problem that gene mutations that can be detected at one time are limited.

Recently, the present inventors have developed a fragment analysis technique using capillary electrophoresis, and have developed an analysis technique that can simultaneously expand the number of gene mutations that can be detected to dozens to hundreds (Fig. 2). In this method, by using a primer 205 in which a double-stranded DNA tag 204 with interstrand cross-linking 203 is linked to a primer portion 205 specific to gene mutation, electric The migration distance can be stably extended to 120 bp or more. It becomes possible to utilize an electrophoretic region that could not be effectively used until now, and at the same time, it is possible to increase the number of gene mutations that can be detected. Fragment analysis using capillary electrophoresis can well separate nucleic acids up to about 600 bases, and can increase the number of available gene mutations. As in the conventional method, gene mutation such as SNP is achieved by attaching ddNTPs modified with four types of fluorescent dyes to the 3' end of a primer linked to a double-stranded DNA tag that is crosslinked between strands by a single-base extension reaction. to detect

During this development process, the inventors attempted to further improve the above method. After addition of a fluorescent dye to the 3' end by single base extension reaction, unreacted fluorescent dye is added in addition to the target product, the primer with a fluorescent dye added to the 3' end by single base extension reaction. no primer remains. The existence of this residual primer may lead to a decrease in detection sensitivity of gene mutations in the fragment analysis method by capillary electrophoresis. In fragment analysis using capillary electrophoresis, nucleic acid samples are injected by electrokinetic injection using electrophoresis (Fig. 3). In this injection method, the negatively charged nucleic acid sample is also injected into the capillary. The remaining primer will be injected into the capillary as well. As a result, the concentration of the end-modified primer, which is the target product injected into the capillary, is relatively decreased, possibly leading to a decrease in detection sensitivity.

The presence of this residual primer has a greater influence as the mutation rate of gene mutations to be detected is lower. In order to improve the detection efficiency for gene mutations with a low mutation rate, it is necessary to increase the primer concentration in order to increase the reaction efficiency of the single-base extension reaction, but high primer concentrations lead to an increase in residual primer concentration. , leading to a lower injection ratio of the target product, the end-modified primer, during sample injection.

Therefore, in the fragment analysis method by capillary electrophoresis, in order to detect target genes (for example, tumor-derived genes) with higher sensitivity, it is necessary to remove unreacted residual primers after the single-base extension reaction and obtain the target product. It is desirable to select and purify certain end-modified primers. However, the only difference between the end-modified primers, which are the target products, and the remaining unreacted primers is the ddNTP with a fluorescent dye attached to the 3' end, and it is necessary to select and purify using the presence or absence of this modification. There is However, it has been difficult to selectively remove only unreacted residual primers by conventional methods known so far.

In a method for detecting gene mutations using fragment analysis by capillary electrophoresis, the only difference between the two is the presence or absence of terminally modified molecules in a solution in which terminally modified selective primers and unreacted residual primers coexist. is. Accordingly, an object of the present invention is to selectively purify a primer having a modified molecule at its end to improve the sensitivity of gene analysis or improve the detection efficiency of gene mutation.

As a result of investigations to solve the above problems, in the above-mentioned fragment analysis method by electrophoresis, an exonuclease that is active even under heat denaturation conditions dissociates the three-dimensional structure of the nucleic acid at the heat denaturation temperature, We have found that by selectively removing only unreacted residual primers, it is possible to purify nucleic acids having modified molecules at their ends. In addition, when a single-base extension reaction is performed by binding a double-stranded DNA tag with interstrand cross-linking to the primer, after dissociating the inter-strand cross-linking, exonuclease treatment is performed to similarly modify the end with a modified molecule. We also obtained the knowledge that it is possible to purify a nucleic acid having The present invention was completed based on the above findings.

Therefore, in one aspect, the present invention provides a method for purifying a target nucleic acid having a terminal modification molecule in a method for detecting a target nucleic acid or determining the base of a target nucleic acid based on the mobility of the nucleic acid in electrophoresis, comprising: ,
A double-stranded nucleic acid having a modified molecule at the end of one of the nucleic acid strands is subjected to a heat denaturation treatment to form a single-stranded nucleic acid,
A method is provided comprising treating the resulting single-stranded nucleic acid with an exonuclease at a heat denaturation temperature to remove nucleic acids other than the nucleic acid strands having terminal modification molecules.

In another aspect, the invention provides a method of detecting the presence of a target nucleic acid and/or determining the base of a target nucleic acid in a sample, comprising:
preparing a sample containing or suspected of containing the target nucleic acid;
preparing a primer comprising a double-stranded nucleic acid tag having an interstrand bridge and a primer nucleic acid that specifically binds to the target nucleic acid;
Performing a single-base extension reaction using the target nucleic acid as a template and the primer,
dissociating interstrand crosslinks in the double-stranded nucleic acid tag;
The double-stranded nucleic acid tag and the double-stranded nucleic acid of the target nucleic acid and the primer are converted to single-stranded nucleic acids by thermal denaturation,
The resulting single-stranded nucleic acid is treated with an exonuclease at a heat denaturation temperature to remove nucleic acids other than the nucleic acid strand having the modified molecule at the end,
A method is provided comprising subjecting the resulting reaction product to capillary electrophoresis for analysis.

In a further aspect, the present invention provides a kit for purifying a target nucleic acid having a terminal modification molecule in a method for detecting a target nucleic acid or determining the base of a target nucleic acid based on the mobility of the nucleic acid in electrophoresis, , an exonuclease.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to selectively purify primers (nucleic acids) having modified molecules at their ends by selectively removing only unreacted residual primers in a fragment analysis method using electrophoresis. The presence of the modified molecule at the 3-terminus inhibits nucleolysis by single-stranded DNA-specific exonucleases in the 3'→5' direction, leaving the terminal-modified selective primer undegraded. On the other hand, the remaining unreacted primer is nucleolyzed from the 3-terminal direction, and by using an exonuclease that is active even under heat denaturation conditions, inhibition of decomposition due to conformation is suppressed. When interstrand-crosslinked double-stranded DNA tags are ligated, the degradation inhibition by exonuclease can be suppressed by dissociating the crosslinks in advance. Therefore, according to the present invention, it becomes possible to improve the sensitivity of genetic analysis or improve the detection efficiency for gene mutations.

Problems, configurations, and effects other than those described above will be clarified by the following description of the embodiments.

1 is a schematic diagram of a fragment analysis technique using capillary electrophoresis; FIG. Schematic representation of a fragment analysis technique using capillary electrophoresis using selective primers ligated with interstrand cross-linked double-stranded nucleic acid tags. It is a schematic diagram explaining an electrokinetic injection method. FIG. 2 is a conceptual diagram of unreacted residual primers and primers having modified molecules at their ends. 1 is a schematic diagram of an example of a selective purification flow for primers with modified molecules at their ends. FIG. The structure of the selective primer linked with the interstrand cross-linking double-stranded DNA tag used in the Examples is shown.

BEST MODE FOR CARRYING OUT THE INVENTION An embodiment of the present invention will be described below based on the drawings. Although the accompanying drawings show specific embodiments consistent with the principles of the invention, they are for the purpose of understanding the invention and are by no means intended to be used as a limiting interpretation of the invention. It is not something that can be done.

The present invention provides a method and kit for purifying a target nucleic acid having a modified molecule at its end, and such a purification method, in a method for detecting a target nucleic acid or determining the base of a target nucleic acid based on the mobility of the nucleic acid in electrophoresis. It relates to a method for detecting a target nucleic acid or determining the base of a target nucleic acid using

4 and 5 for a method of selectively purifying only primers having modified molecules at their ends by conducting a single-base extension reaction using selective primers bound with double-stranded nucleic acid tags having interstrand cross-links. be used to explain. However, the tag labels of the selective primers are not limited to the interstrand-crosslinked double-stranded nucleic acid tags shown in FIGS. can be used.

FIG. 4 shows a primer 400 having a modified molecule at the end and a remaining unreacted primer 406, which are generated by the reaction step of adding a modified molecule to the end in the fragment analysis method using capillary electrophoresis shown in FIG. show. Here, selective primer portion 405 has complementary sequences that hybridize to nucleic acid sequences having specific genetic mutations. A nucleic acid tag 404 is linked to the primers 400 and 406 to adjust electrophoretic mobility. In FIG. 4, the nucleic acid tag 404 for adjusting electrophoretic mobility has a double-stranded nucleic acid structure with interstrand crosslinks.

FIG. 5 shows an example flow of selective purification of primers having modified molecules at their ends. First, a selective primer is prepared. In FIG. 5, primers are used that are linked to single-stranded nucleic acid regions that act as selective primers linked to double-stranded nucleic acid tags with interstrand bridges. A ddNTP having a modified molecule (for example, fluorescence) corresponding to a specific base is added to the 3' end of the single-stranded region of the primer by a single-base extension reaction using this primer. At this time, ddNTPs having modified molecules are not given to all primers, and unreacted primers also remain in the solution ((1) in FIG. 5). The solution in which the primers having modified molecules at their ends and the unreacted residual primers coexist is subjected to the following treatment.

First, as shown in FIG. 5, when a double-stranded nucleic acid tag having an interstrand crosslink is linked to a selective primer, a step of dissociating the crosslinked structure is performed to dissociate the crosslinked structure between the double strands ( (2) in Fig. 5). Typically, cross-linking between chains is performed by photocrosslinking, and therefore, the cross-linking between chains is dissociated by irradiation with ultraviolet light having a wavelength for dissociating cross-links. Note that this step is not necessary if a double-stranded nucleic acid tag with interstrand crosslinks is not used.

Subsequently, the nucleic acid molecule is decomposed using a heat-stable exonuclease while being thermally annealed ((3) in FIG. 5). Here, the exonuclease is a single-stranded DNA-specific exonuclease in the 3′→5′ direction. As will be described later, the presence of ddNTPs at the 3' ends suppresses the degradation of primers having modified molecules at their ends, but all other nucleic acid molecules that have become single-stranded due to thermal annealing are 3' Disassembled from the end. At this time, the thermal annealing treatment not only dissociates the double-stranded structure, but also has the effect of suppressing the formation of the three-dimensional structure of the single-stranded nucleic acid. Therefore, the exonuclease used in the present invention is preferably an exonuclease that is active even at thermal annealing temperatures. Through such steps, it becomes possible to selectively purify a primer having a modified molecule at its end ((4) in FIG. 5).

Therefore, in one aspect, the present invention provides a method for purifying a target nucleic acid having a terminal modification molecule in a method for detecting a target nucleic acid or determining the base of a target nucleic acid based on the mobility of the nucleic acid in electrophoresis, comprising: ,
A double-stranded nucleic acid having a modified molecule at the end of one of the nucleic acid strands is subjected to a heat denaturation treatment to form a single-stranded nucleic acid,
A method is provided comprising treating the resulting single-stranded nucleic acid with an exonuclease at a heat denaturation temperature to remove nucleic acids other than the nucleic acid strands having terminal modification molecules.

As used herein, a “target nucleic acid having a terminal modification molecule” is a primer into which a terminal modification molecule has been incorporated by a single-base extension reaction. Here, the terminus is preferably the 3' terminus. Alternatively, a labeled dideoxynucleotide (ddNTP), such as a fluorescently labeled ddNTP, can be used as the modifying molecule.

A double-stranded nucleic acid having a modified molecule at the end of one of the nucleic acid strands by the single-base extension reaction (that is, a selective primer with a modified molecule incorporated at the end by the single-base extension reaction and a target nucleic acid that serves as a template). double-stranded nucleic acid). This double-stranded nucleic acid is converted to a single-stranded nucleic acid by thermal denaturation. As for the heat denaturation treatment conditions, a person skilled in the art can set appropriate temperature conditions, buffer conditions and the like based on the GC content of the designed selective primer. For example, the heat denaturation treatment conditions can be 60 to 100°C, for example, under a heat denaturation temperature of about 85°C.

The resulting single-stranded nucleic acid is then treated with an exonuclease at a heat denaturation temperature. The heat denaturation temperature may be any temperature that can suppress annealing of single-stranded nucleic acids to form double strands, and may be the same as the heat denaturation treatment conditions described above, or may be different. may be the condition of Preferably, the heat denaturation temperature is the optimum temperature for the exonuclease used. The exonuclease has a 3'→5' single-stranded nucleic acid-specific exonuclease activity and is preferably a thermostable exonuclease.

Examples of such exonucleases include, but are not limited to:
・TK1646 from the hyperthermophilic archaea Thermococcus kodakarensis KOD1 (Saeed, MS and Rashid, N., International Journal of Biological Macromolecules, 140:1194-1201, 2019)
・PhuExo I from the hyperthermophilic archaea Pyrococcus furiosus (Tori et al., PLOS One, 8:e58497, 2013)
・PhoExo I from the hyperthermophilic archaea Pyrococcus horikoshii (Miyazono et al., Acta Crystallogr. F. Struct. Bio. Commun., 70:1076-1079, 2014)
TK1646 is a 3'→5' single-stranded DNA-specific exonuclease that is active only at 80-100°C, and PhuExo I and PhoExo I are known to be active even at 65°C. and is suitable as an exonuclease in the method. Therefore, in one embodiment, it is preferable to use at least one selected from the group consisting of TK1646, PhuExo I, and PhoExo I as the exonuclease.

In addition, ddNTP, which is a typical molecule as a modified molecule at the end, has been reported to inhibit the activity of nucleases and exonucleases (catalog NEB Expressions Issue II 2019, literature J.W. Hanes and K.A. Johnson, Antimicrobial Agents and Chemotherapy, 52:253-258, 2008). In this way, nucleic acids having ddNTPs at their 3-terminals can be inhibited from being degraded by exonucleases in the 3'→5' direction.

Exonuclease treatment removes nucleic acids other than nucleic acid strands with modified molecules at their ends. Therefore, according to this method, it becomes possible to selectively recover and purify a target nucleic acid having a modified molecule at its end.

In one embodiment, a double-stranded nucleic acid having a modification molecule at the end of one nucleic acid strand obtained by a single-base extension reaction may contain a double-stranded nucleic acid tag crosslinked between strands. The details of this interstrand-crosslinked double-stranded nucleic acid tag will be described later. In this case, the method further comprises dissociating the interstrand bridges in the double-stranded nucleic acid tag. Preferably, interstrand crosslinks are dissociated prior to heat denaturation of the double-stranded nucleic acid. For example, when the interstrand cross-linking is due to photo-crosslinking, the inter-strand cross-linking can be dissociated by a means for dissociating photo-crosslinks, for example, irradiation with light of a specific wavelength. Dissociation of interstrand crosslinks can be carried out as appropriate, depending on the type of crosslinks used.

In another aspect, the invention provides a method of detecting the presence of a target nucleic acid and/or determining the base of a target nucleic acid in a sample, comprising:
preparing a sample containing or suspected of containing the target nucleic acid;
preparing a primer comprising a double-stranded nucleic acid tag having an interstrand bridge and a primer nucleic acid that specifically binds to the target nucleic acid;
Performing a single-base extension reaction using the target nucleic acid as a template and the primer,
dissociating interstrand crosslinks in the double-stranded nucleic acid tag;
The double-stranded nucleic acid tag and the double-stranded nucleic acid of the target nucleic acid and the primer are converted to single-stranded nucleic acids by thermal denaturation,
The resulting single-stranded nucleic acid is treated with an exonuclease at a heat denaturation temperature to remove nucleic acids other than the nucleic acid strand having the modified molecule at the end,
A method is provided comprising subjecting the resulting reaction product to capillary electrophoresis for analysis.

A case where the purification method according to the present invention described above is applied to a gene analysis method will be described.
First, a sample containing or suspected of containing a target nucleic acid is prepared. The sample is not particularly limited as long as it contains nucleic acids, and can be any of biological samples (e.g., cell samples, tissue samples, liquid samples, etc.) and synthetic samples (e.g., nucleic acid libraries such as cDNA libraries). A sample can be used. In the case of samples derived from living organisms, the organism from which the sample is derived is not particularly limited, and may be vertebrates (e.g., mammals, birds, reptiles, fish, amphibians, etc.), invertebrates (e.g., insects, nematodes, crustaceans, etc.). ), protist, plant, fungus, bacterium, virus, etc., can be used. For example, when assuming cancer testing in humans, nucleic acid-containing samples obtained from humans to be tested, such as whole blood, serum, plasma, saliva, urine, feces, skin tissue, and cancer tissue, are prepared.

The target nucleic acid is not particularly limited as long as it contains a sequence to be detected or a base to be determined. Deoxyribonucleic acid (DNA) such as genomic DNA, cDNA, and ribonucleic acid (RNA) such as messenger RNA (mRNA), as well as fragments thereof, are included. In the present invention, it is preferable to use, for example, cell-free DNA (cfDNA, DNA that is free in blood) and circulating tumor DNA (ctDNA) as the target nucleic acid. Preparation of nucleic acids from a sample can be performed by methods known in the art. For example, when preparing target nucleic acids from blood or cells, proteolytic enzymes such as proteinase K, chaotropic salts such as guanidine thiocyanate and guanidine hydrochloride, surfactants such as Tween and SDS, or commercially available cell lysis reagents are used. It can be used to lyse cells and elute the nucleic acids contained therein, ie DNA and RNA. When RNA is prepared, among the nucleic acids eluted by cell lysis, DNA is degraded with DNase to obtain a sample containing only RNA as nucleic acid. When preparing mRNA, since mRNA contains a poly-A sequence, it is possible to capture only mRNA from an RNA sample prepared as described above using a DNA probe containing a poly-T sequence. Kits for such nucleic acid preparation are sold by many manufacturers, and it is possible to simply purify the target nucleic acid.

A primer is also prepared that includes a double-stranded nucleic acid tag with an interstrand crosslink and a primer nucleic acid that specifically binds to a target nucleic acid. The double-stranded nucleic acid tag has a mobility-distinguishable length, and the primer nucleic acid is designed to bind specifically to the target nucleic acid to produce a single-base extension reaction.

A double-stranded nucleic acid tag having interstrand cross-links may be a DNA, RNA, or hybrid nucleic acid, as long as the nucleic acid has a double-stranded nucleic acid structure. Preferably, the double-stranded nucleic acid is double-stranded DNA.

A double-stranded nucleic acid tag has at least one interstrand crosslink. In the present invention, "interstrand cross-linking" means that one strand and the other strand of a double-stranded nucleic acid are cross-linked at at least one site. A method for intramolecular cross-linking between two chains is not particularly limited as long as it is a method known in the art. Preferably, the interstrand cross-linking is by photocrosslinking.

Cross-linking molecules such as classically known nitrogen mustard, cisplatin, carmustine, mitomycin C, psoralen, trioxane (trimethylpsoralen), malondialdehyde, etc. can be used for interstrand cross-linking (e.g. Guainazzi et al., Cellular and Molecular Life Sciences, 67:3683-3697, 2010). These cross-linking molecules are of the type in which one cross-linking molecule enters between bases, and since it enters between A and T or between G and C, the cross-linking position is random when viewed as a whole nucleic acid molecule, and the cross-linking efficiency is 30-40%. degree. For example, psoralen is a photocrosslinker that produces photocrosslinks at the 5'-TA-3' sequence by photoreaction at a photoligation wavelength of 350 nm, and cleaves the crosslinks at a photocleavage wavelength of 250 nm.

In addition, CNV-K (molecular name: 5'-O-(4,4'-Dimethoxytrityl)-1'-(3-cyanovinylcarbazol-9-yl )-2'-deoxy-β-D-ribofuranosyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), CNV-D (molecular name: 3-O-(4,4 '-Dimethoxytrityl)-2-N-(N-carboxy-3-cyanovinylcarbazol)-D-threonin-1-yl-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) etc. (For example, Patent No. 4940311, Yoshimura et al., ChemBioChem, 10:1473-1476, 2009, Sakamoto et al., Org. Lett., 17:936-939, 2015). These molecules undergo a [2+2] circularization reaction with the pyrimidine base (thymine, cytosine, or uracil) of the complementary strand at a position shifted by one base, starting with the irradiation of ultraviolet light (366 nm) as a reaction trigger. A cross-linking point can be formed by this. Moreover, since it can be introduced into the backbone of a nucleic acid molecule, it is practically preferable that the cross-linking point can be arbitrarily designed. It is also important from a practical point of view that these photocrosslinking molecules can reversibly dissociate the interchain crosslinks by exciting them with ultraviolet light of a different wavelength (312 nm). Photocrosslinking molecules CNV-K and CNV-D are particularly suitable from the standpoint of availability, cost and the like.

Another cross-linking molecule may be a cross-linking molecule using a Click reaction with a combination of an azide group ₍ -N3) and an alkyne group. Such cross-linking points are reported, for example, in Kocalta et al., ChemBioChem, 9:1280-1285, 2008. As a cross-linking molecule corresponding to a specific base sequence, for example, UTA-6026, which is specific to the sequence 5'-CAATTA-3'/3'-GTTAAT-5' and bridges AGs separated by 5 bases, is known (Zhou et al., J. Am. Chem. Soc., 123:4865-4866, 2001). Another is ImImPy, which is specific for the sequence 5′-Py(T/C)GGC(T/A)GCCPu(A/G)-3′ and bridges between bases 9 bases apart (Bando et al., J. Am Chem. Soc., 123:5158-5159, 2001) and a C8/C8′-tripyrrole-linked sequence-selective pyrrolo that is specific for the sequence 5′-GCTTATAATGG-3′ and bridges between bases separated by 11 bases[2]. ,1-c][1,4]benzodiazepine (PBD) dimer (Tiberghien et al., Bioorganic & Medicinal Chemistry Letters, 18:2073-2077, 2008) and the like are known. An advantage of these sequence-specific cross-linking molecules is that the cross-linking points can be designed.

A double-stranded nucleic acid tag with interstrand cross-links defines a migration distance (mobility) in electrophoresis. That is, by linking double-stranded nucleic acid tags of different lengths to primers, migration distances can be altered in electrophoresis. Capillary electrophoresis can detect nucleic acids with a chain length of up to about 600 bases. Stranded nucleic acid tags can range in length from 1 to about 590 bases in length.

The base sequence of the double-stranded nucleic acid tag is not particularly limited as long as it is a nucleic acid having interstrand crosslinks. Also, the double-stranded nucleic acid tag can be chemically synthesized by a known oligonucleotide synthesis technique, but is usually synthesized using a commercially available chemical synthesizer.

A primer nucleic acid that specifically binds to a target nucleic acid (also referred to herein as a selective primer) may be either DNA or RNA, and the type of target nucleic acid and the type of polymerase used for the single-base extension reaction. determined according to Preferably, the primer nucleic acid is DNA, and a single-base extension reaction is performed using DNA or mRNA as the target nucleic acid as a template.

A primer nucleic acid is designed to have a sequence that specifically binds to a target nucleic acid (or target region), ie, has a sequence that is complementary to the target nucleic acid (or target region). Techniques for designing primers are well known in the art, and primers that can be used in the present invention satisfy conditions that allow specific annealing, such as length and base composition (melting temperature) that allow specific annealing designed to have For example, the length that functions as a primer is preferably 10 bases or more, more preferably 15 to 50 bases, still more preferably 15 to 30 bases, such as about 20 bases. In designing, it is preferable to confirm the GC content of the primer and the melting temperature (Tm) of the primer. Tm means the temperature at which 50% of any nucleic acid strand hybridizes with its complementary strand. needs to be optimized. On the other hand, if the temperature is too low, non-specific reactions will occur, so the temperature should be as high as possible. Known primer design software can be used to confirm the Tm. The designed primer can be chemically synthesized by a known oligonucleotide synthesis technique, but is usually synthesized using a commercially available chemical synthesizer.

The primers used in this method, which include interstrand-crosslinking double-stranded nucleic acid tags and selective primers, can be attached by any method. For example, after preparing a sequence in which one strand of a double-stranded nucleic acid tag and a selective primer are bound directly or via a spacer, the other strand of the double-stranded nucleic acid tag is annealed to form a double-stranded nucleic acid portion. A primer can be prepared by forming an interstrand crosslink in at least one location. Alternatively, a double-stranded nucleic acid tag with interstrand cross-links may be prepared and then attached to a selective primer either directly or via a spacer. The ligation method may be hydrogen bonding based on base sequence complementarity, or ligation may be performed using a known ligase.

In one embodiment, the method uses multiple primers comprising double-stranded nucleic acid tags of different lengths and primer nucleic acids that specifically bind to different target nucleic acids. As noted above, the base length of a double-stranded nucleic acid tag that provides a discernible migration distance is 1 base. For example, double-stranded nucleic acid tags differing in length by 5 bases or more, preferably 10 bases or more are bound to different primers. In this method, for example, from 1 to about 100 different target nucleic acids can be detected simultaneously.

Subsequently, a single-base extension reaction is performed using the target nucleic acid as a template and a primer. Single-base extension reactions are known in the art and typically are single-base extension reactions using a polymerase. The polymerase to be used is selected according to the type of template (target nucleic acid) and the type of primer to be used. For example, a DNA-dependent or RNA-dependent DNA polymerase is used for a single-base extension reaction using a DNA primer with DNA or RNA as a template, respectively.

Single-base elongation reactions are widely known in the art, and non-patent document 3, for example, describes a method for efficiently elongating one base by a cycle reaction.

When a target nucleic acid is present, a selective primer that specifically binds to this target nucleic acid is hybridized, and a base is incorporated as a substrate from the 3' end portion of the selective primer by a polymerase synthesis reaction. At this time, by using, for example, a dideoxynucleotide (ddNTP) as a base (substrate) to be incorporated, the synthesis reaction is completed with only one-base elongation. In one embodiment, a single-base extension reaction is performed using a modified molecule, eg, a labeled ddNTP, as a substrate. A label is useful for conveniently detecting whether it has been incorporated or for determining the type of incorporated base, and any label known in the art can be used. Such labels include radioactive isotopes ( ³² P, ¹²⁵ I, ³⁵ S, etc.), fluorescent substances, luminescent substances (luciferin, etc.), and the like. Fluorescent substances can preferably be used, including but not limited to fluorescein (FITC), sulforhodamine (TR), tetramethylrhodamine (TRITC), carboxy-X-rhodamine (ROX), carboxytetramethylrhodamine (TAMRA), NED, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5'-hexachlorofluorescein CE-phosphoramidite (HEX), 6-carboxy-4',5'- Dichloro-2',7'-dimethoxyfluorescein (JOE), 5'-tetrachlorofluorescein CE-phosphoramidite (TET), Rhodamine 110 (R110), Rhodamine 6G (R6G), VIC®, ATTO series, dR110 (carboxy-dichloro rhodamine 110), dR6G (dihydro rhodamine 6G), dTAMRA (Tetramethyl rhodamine), dROX (carboxy- X-rhodamine) and the like. For example, when trying to determine the type of base, it is excited and detected at different wavelengths to distinguish between 4 types of bases and 5 types for reference (to detect and correct base length from reference ladder DNA). Five types of fluorescent substances can be used in combination. There are no particular restrictions on the type of such label, the method of introducing the label, and the like, and conventionally known various means can be used. In a preferred embodiment, fluorescently labeled dideoxynucleotides (ddNTPs) are used as modifying molecules.

The presence or absence of the target nucleic acid can be determined by whether or not this single-base extension occurs, and the specific base in the target nucleic acid can be determined based on the type of base incorporated in the single-base extension portion. becomes. For example, when the purpose is to detect a single nucleotide polymorphism (SNP), a selective primer that specifically binds to the upstream portion of the SNP is designed, hybridized with the target nucleic acid, and labeled with a different label. A single-base extension reaction is performed using a base (modifying molecule) having as a substrate. The SNP of the target nucleic acid can be detected by determining the type of incorporated base based on the label.

After the single-base extension reaction, the above-described method for purifying the target nucleic acid according to the present invention is performed. Specifically, the interstrand crosslinks in the double-stranded nucleic acid are dissociated, and the double-stranded nucleic acid and the double-stranded nucleic acid of the target nucleic acid and the primer are converted to single-stranded nucleic acids by heat denaturation treatment, and the resulting single-stranded nucleic acid Nucleic acids are treated with an exonuclease under a heat denaturation temperature to remove nucleic acids other than the nucleic acid strands having terminal modification molecules. These steps can be performed as described above.

The resulting reaction is then analyzed by capillary electrophoresis (CE). CE is a technique that separates introduced components by differences in mobility based on charge, size, shape, and the like. In this method, the target nucleic acid of interest is detected from the type of target nucleic acid based on mobility and the presence or absence of the target nucleic acid or the type of specific bases in the target nucleic acid based on the modified molecules incorporated in the single-base extension reaction, and/or bases of the target nucleic acid can be determined.

As described above, in the present invention, exonucleases are used to purify target nucleic acids having modified molecules at their ends, and to perform genetic analysis with high sensitivity and high efficiency. The method according to the present invention can be performed simply and rapidly, for example, by using a kit containing exonuclease.

Therefore, in a further aspect, the present invention provides a kit for purifying a target nucleic acid having terminal modification molecules in a method for detecting a target nucleic acid or determining the base of a target nucleic acid based on the mobility of the nucleic acid in electrophoresis. provided, such kits include an exonuclease. In one embodiment, the exonuclease is a 3' to 5' single-stranded nucleic acid specific exonuclease. Also in preferred embodiments, the exonuclease comprises at least one selected from the group consisting of a thermostable exonuclease, eg, TK1646, PhuExoI, and PhoExoI.

In addition to exonuclease, this kit contains buffers that make up the reaction solution, double-stranded nucleic acid tags with interstrand cross-links, modified molecules (labeled dNTPs or ddNTP mixtures), enzymes (polymerase, reverse transcriptase, etc.) , a standard sample for calibration, an instruction manual describing the procedure for use, the amount of use, and the like. The kit may be provided ready-to-use or non-ready-to-use. Such forms and preparations can be understood by those skilled in the art.

[Example]
Specific examples will be described below.

(1) Preparation of selective primer with interstrand cross-linked double-stranded DNA tag linked Inter-strand cross-linked double-stranded DNA tag for fragment analysis technique using capillary electrophoresis CNV-D was employed as a bridging molecule in selective primers.

Table 1 shows oligonucleotide sequences (manufactured by Hokkaido System Science Co., Ltd.) as model samples. As shown in Fig. 6, photocrosslinks (indicated by white diamonds in the figure) were formed between Oligo 1 and Oligo 2, and between Oligo 2 and Oligo 3, and nicks (dotted lines in the figure) were formed between Oligo 1 and Oligo 3. ) are linked by ligation. Here, oligo 1 and oligo 2 are interstrand-crosslinked double-stranded DNA tags that modulate electrophoretic mobility, and oligo 3 serves as a selective primer to specific genetic mutations. In particular, Oligo 3 is designed to detect KRAS G12 gene mutations (G12D, G12V, G12A), and the 3' terminal 28 bases of Oligo 3 are designed to specifically bind to genes. there is

First, each oligo was mixed with a buffer solution (1x PCR buffer for KOD-Plus Neo, 2.5 mM MgSO ₄ , manufactured by Toyobo) at an equal ratio of 1:1:1 so that the final concentration was 25 μM, and incubated at 95°C for 2 minutes. →Three types of oligos were hybridized in a temperature step of 20 minutes at room temperature and 20 minutes at 4°C. Then, using T4 Ligase (manufactured by New England Biolab), reaction was carried out at room temperature for 30 minutes to ligate Oligo 1 and Oligo 3.

Next, ultraviolet light (wavelength: 366 nm, irradiation time: 1 seconds) to form photocrosslinks between the double-stranded DNAs. The obtained selective primers to which the interstrand-crosslinked double-stranded DNA shown in FIG. The separated band was excised from the gel, dissolved with β-Agarase (Thermostable β-Agarase, manufactured by Nippon Gene), and then purified by ethanol precipitation.

(2) Single-base extension reaction using selective primer Using the obtained selective primer (input amount: 0.1 pmol) to which the interstrand-crosslinked double-stranded DNA tag is linked, template DNA (input amount: 100 pmol/μL), a DNA polymerase capable of incorporating ddNTPs with a fluorescent dye (Therminator DNA Polymerase, New England Biolab), four types of ddNTPs with a fluorescent dye (final concentration 1 μM, R6G-ddATP, ROX-ddUTP, TAMRA-ddCTP, R110-ddGTP, manufactured by Perkin Elmer), 1-base extension reaction was performed with 40 cycles of 96°C for 10 seconds → 50°C for 5 seconds → 60°C for 30 seconds.

(3) Treatment with heat-stable 3′→5′-degrading exonuclease After that, photocrosslinks between double-stranded DNAs were dissociated by irradiation with ultraviolet light (312 nm, 2 minutes). Next, TK1646, a thermostable 3′→5′-degrading exonuclease, and Mn ²⁺ as a cofactor were added, and heat-treated at 85° C. in a solution of 10 μg BSA 1 mM MnCl ₂ 25 mM Tris-HCl (pH 9.0). The exonuclease reaction was carried out for 30 minutes under denaturing temperature. 0.5 μL of the obtained treatment solution, 0.5 μL of size standard GeneScan-120LIZ and 9 μL of Hi-Diformamide were mixed and denatured at 95° C. for 5 minutes.

The finally obtained sample was measured with a capillary electrophoresis device (SeqStudio Genetic Analyzer, manufactured by Thermo Fisher Scientific). From the results of fragment analysis obtained, it was confirmed that the peak intensity increased after exonuclease treatment, contributing to the improvement of transfection efficiency during electrokinetic injection.

The present invention is not limited to the embodiments and examples described above, but includes various modifications. For example, the above-described embodiments and examples have been described in detail in order to explain the present invention in an easy-to-understand manner, and do not necessarily include all the configurations described. Also, part of one embodiment or example can be replaced with the configuration of another embodiment or example. Moreover, the configuration of another embodiment or example can be added to the configuration of one embodiment or example. In addition, a part of the configuration of each embodiment or each example can be added, deleted or replaced with a part of the configuration of another embodiment or example.

100…selective primer
101, 201, 401...Template molecules
102, 202, 402... Modifier molecules
200 Primer
203 ... interstrand cross-linking
204, 404... Double-stranded nucleic acid tags with interstrand crosslinks
205, 405…Selective primer
400...Primer with modification molecule at the end
406…Unreacted residual primer
507 Thermostable single-stranded DNA-specific 3'→5'-degrading exonuclease

SEQ ID NOs: 1-3: artificial (synthetic oligonucleotides)

Claims (15)

A method for detecting a target nucleic acid or determining the base of a target nucleic acid based on the mobility of the nucleic acid in electrophoresis, wherein a target nucleic acid having a terminal modification molecule is purified, comprising:
A double-stranded nucleic acid having a modified molecule at the end of one of the nucleic acid strands is subjected to a heat denaturation treatment to form a single-stranded nucleic acid,
A method comprising treating the resulting single-stranded nucleic acid with an exonuclease at a heat denaturation temperature to remove nucleic acids other than the nucleic acid strands having terminal modification molecules. 2. The method of claim 1, wherein the double-stranded nucleic acid comprises an interstrand-crosslinked double-stranded nucleic acid tag. 3. The method of claim 2, further comprising dissociating interstrand bridges in said double stranded nucleic acid tag. A method of detecting the presence of a target nucleic acid and/or determining the base of a target nucleic acid in a sample, comprising:
preparing a sample containing or suspected of containing the target nucleic acid;
preparing a primer comprising a double-stranded nucleic acid tag having an interstrand bridge and a primer nucleic acid that specifically binds to the target nucleic acid;
Performing a single-base extension reaction using the target nucleic acid as a template and the primer,
dissociating interstrand crosslinks in the double-stranded nucleic acid tag;
The double-stranded nucleic acid tag and the double-stranded nucleic acid of the target nucleic acid and the primer are converted to single-stranded nucleic acids by thermal denaturation,
The resulting single-stranded nucleic acid is treated with an exonuclease at a heat denaturation temperature to remove nucleic acids other than the nucleic acid strand having the modified molecule at the end,
A method comprising subjecting the resulting reaction product to capillary electrophoresis for analysis. 5. The method of claim 4, wherein the single-base extension reaction is performed using the modifying molecule as a substrate. 6. The method of claim 1 or 5, wherein the modifying molecule comprises a fluorescently labeled dideoxynucleotide (ddNTP). 5. The method of claim 1 or 4, wherein said end is the 3' end. 5. The method of claim 1 or 4, wherein the exonuclease is a 3' to 5' single-stranded nucleic acid-specific exonuclease. 5. The method of claim 1 or 4, wherein said exonuclease is a thermostable exonuclease. 10. The method of claim 9, wherein the thermostable exonuclease comprises at least one selected from the group consisting of TK1646, PhuExoI, and PhoExoI. A method for detecting a target nucleic acid or determining the base of a target nucleic acid based on the mobility of the nucleic acid in electrophoresis, wherein the kit is for purifying a target nucleic acid having a modified molecule at the end thereof, the kit comprising an exonuclease. Kit to be. 12. The kit of claim 11, wherein the exonuclease is a 3' to 5' single-stranded nucleic acid-specific exonuclease. 12. The kit of claim 11, wherein said exonuclease is a thermostable exonuclease. 14. The kit according to claim 13, wherein the thermostable exonuclease comprises at least one selected from the group consisting of TK1646, PhuExoI, and PhoExoI. 12. The kit of claim 11, further comprising at least one component selected from the group consisting of double-stranded nucleic acid tags with interstrand crosslinks, polymerases, and modifying molecules.

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