Labeling of nucleic acid molecules with interstrand cross-linked double-stranded DNA

Abstract

To provide a method and means for improving a single base extension reaction method used in capillary electrophoresis.SOLUTION: The present invention pertains to a method for detecting the presence of a target nucleic acid in a sample and / or for determining a base in the target nucleic acid. The method comprises: preparing a sample including or being suspected to include a target nucleic acid; preparing a primer 200 including a double-strand nucleic acid tag 204 that has an interstrand crosslink 203, and a primer nucleic acid 205 that specifically binds to the target nucleic acid; performing a single base extension reaction using the primer with the target nucleic acid as a template; and analyzing the obtained reactant by subjecting the same to capillary electrophoresis.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to methods and means for genetic analysis based on electrophoretic mobility in the measurement of biomolecules, particularly nucleic acid molecules. [Background technology]

[0002] There are various individual differences in genomes, and these differences in genome sequence are useful biomarkers as indicators of disease and drug response. Genomic mutations are mainly detected by PCR detection, base sequence analysis using a sequencer, and single-base extension reaction analysis (Patent Document 1).

[0003] Recent advances in genomic science have made it possible to perform panel testing for genome mutations using massively parallel sequencers, which are capable of large-scale analysis. Simultaneous analysis of multiple gene mutations makes it possible to simultaneously determine many diseases and treatment options. In particular, liquid biopsy-based cancer screening is expected to see significant advances in the future. Because liquid biopsy is a blood-based test, it is minimally invasive and can be used to test for cancer throughout the body, leading to ongoing research into its potential as a new form of cancer screening (Non-Patent Document 1). However, the cost of analysis poses a challenge when considering the practical implementation of these liquid biopsy-based cancer screening technologies. The development of low-cost multi-mutation testing technologies that can replace expensive massively parallel sequencers is necessary for the practical implementation of liquid biopsy-based cancer screening. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 5,888,819 [Non-patent literature]

[0005] [Non-Patent Document 1] Cohen, JD et al., Science Vol. 359, pp. 926-930 (2018) [Non-patent document 2] Coutinho, A. et, al., PLOS ONE Volume 9, Issue 3 e93292 (2014) [Non-patent document 3] Dias-Santagata, D. et al., EMBO Molecular Medicine Vol. 2, pp. 146-158 (2010) [Non-patent document 4] Meagher, RJ et al., Anal. Chem. Vol. 79, pp. 1848-1854 (2007) Summary of the Invention [Problem to be solved by the invention]

[0006] One example of a low-cost technology that can detect multiple genetic mutations is fragment analysis using capillary electrophoresis (CE) (Figure 1). This method uses selective primers 100 designed to vary their electrophoretic distance so that each target mutation can be distinguished and to be complementary to the genetic sequence 101 containing the mutation of interest. When a target genetic mutation is present, dideoxynucleotides (ddNTPs) modified with four fluorescent dyes 102 are added to the 3' end of the primer by a polymerase synthesis reaction, exactly corresponding to the genetic mutation (typically a single nucleotide polymorphism, or SNP). This method is called single-base extension. The primers extended by the single-base extension reaction for each genetic mutation with a modified molecule at the end are converted into single-stranded DNA, which is then separated by capillary electrophoresis. The genetic mutation is finally detected by fluorescent detection of the fluorescent dye at the 3' end.

[0007] The fluorescent-labeled single-base extension reaction method uses electrophoretic mobility as an indicator for gene identification. By adjusting the length of the primers used or tagging the primers to alter their mobility, the signal detection position can be varied, enabling simultaneous detection of multiple genes. Coutinho et al. (Non-Patent Document 2) demonstrated simultaneous detection of 26 plexes, while Dias-Santagata et al. (Non-Patent Document 3) simultaneously detected 5–8 mutations, performing this analysis eight times to detect a total of 58 genes. However, the methods described in Non-Patent Documents 2 and 3 only utilize a region of approximately 120 bp within the analytical range of the CE sequencer (Figure 1). While CE sequencers have the precision to resolve single-base differences in length within a range of 50–600 bp, existing fluorescent-labeled single-base extension reaction methods only utilize a portion of the CE sequencer's detection range.

[0008] One of the reasons for the limited detection range of CE sequencers is the difficulty of chemically synthesizing primer oligo DNA of >100 bp or more. Another reason is that as the primer length increases, the long single-stranded sequence portion can cause nonspecific binding during the single-base extension reaction. To solve these problems, mobility correction using labels other than nucleic acids, as in Meagher et al. (Non-Patent Document 4), is effective, but it is not easy to synthesize a large number of these polymers with high purity and polymerization degrees.

[0009] In other words, to expand the capability of the single-base extension reaction method for simultaneous detection of multiple items, it is necessary to be able to synthesize a variety of labeling substances that modify electrophoretic mobility and that do not cause nonspecific binding to primers. Therefore, an object of the present invention is to provide a method and means for improving the single-base extension reaction method in capillary electrophoresis. [Means for solving the problem]

[0010] The present inventors have discovered the usefulness of interstrand-crosslinked double-stranded DNA molecules with various electrophoretic mobilities as primer labels, and have found that by using selective primers to which such interstrand-crosslinked double-stranded DNA molecules are bound, genetic analysis can be performed based on a wider range of mobility differences than previously possible, without causing nonspecific binding, thereby completing the present invention.

[0011] In one aspect, the present invention provides a primer comprising a double-stranded nucleic acid tag having an interstrand bridge and a primer nucleic acid that specifically binds to a target nucleic acid.

[0012] In another aspect, the present invention provides a kit for gene analysis comprising the primers.

[0013] In a further aspect, the present invention provides a primer labeling kit comprising an interstrand bridged double-stranded nucleic acid molecule, the interstrand bridged duplex nucleic acid molecule comprises at least one interstrand bridged duplex nucleic acid unit; the interstrand bridged double-stranded nucleic acid unit is a first oligonucleotide comprising a first base sequence comprising at least one interstrand-bridge-forming base and a second base sequence comprising at least one interstrand-bridge-forming base; a second oligonucleotide comprising a sequence that is complementary to the second base sequence and includes a base that forms an interstrand bridge with the interstrand bridge-forming base in the second base sequence, and a sequence that is complementary to the first base sequence and includes a base that forms a bridge with the interstrand bridge-forming base in the first base sequence; and a primer labeling kit comprising: a first base sequence in a first oligonucleotide and a sequence complementary to the first base sequence in a second oligonucleotide forming a double-stranded nucleic acid.

[0014] In yet another aspect, the present invention provides a method for detecting the presence of a target nucleic acid in a sample and / or determining the base of the target nucleic acid, comprising the steps of: providing a sample containing or suspected of containing a target nucleic acid; providing 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; The resulting reaction product is subjected to capillary electrophoresis for analysis. The present invention provides a method comprising: [Effects of the Invention]

[0015] According to the present invention, by using a double-stranded nucleic acid, which is easy to synthesize in lengths of >100 bp, as a labeling tag for a selective primer, a wider range of detectable strand lengths can be utilized in electrophoresis. Furthermore, by using a double-stranded nucleic acid molecule, which is made inseparable by interstrand crosslinking, as a labeling tag for a selective primer, nonspecific binding between the primer and the labeling tag that are present during the single-base extension reaction is prevented. Therefore, according to the present invention, it is possible to simultaneously detect a larger number of target nucleic acids with high sensitivity. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic diagram of a fragment analysis method using capillary electrophoresis. [Figure 2] FIG. 1 is a schematic diagram of a fragment analysis method using capillary electrophoresis, using selective primers bound to interstrand-bridged double-stranded nucleic acid tags. [Figure 3] The base sequence of the interstrand cross-linked double-stranded DNA used in the interstrand cross-linking test is shown below. [Figure 4] 1 is a photograph showing an electrophoretic image of double-stranded DNA that has been subjected to interstrand cross-linking treatment. [Figure 5] The base sequences of the interstrand cross-linked double-stranded DNA tag and primer portion used in the fluorescent single-base extension reaction test are shown. [Figure 6] 1 is a graph showing the results of capillary electrophoresis (CE) analysis after fluorescent single-base extension reaction using an unlabeled primer (A) or a primer labeled with an interstrand cross-linked double-stranded DNA tag (B). [Figure 7]An example of the base sequence of one unit constituting the tandem structure of an interstrand cross-linked double-stranded DNA tag (A) and an image of the tandem structure formed (B) are shown. [Figure 8] 1 is a photograph showing electrophoretic images of interstrand cross-linked double-stranded DNA tags having tandem structures containing different numbers of units. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is based on the use of interstrand-bridged double-stranded nucleic acid tags to label selective primers in single-base extension reactions in fragment analysis techniques using capillary electrophoresis.

[0018] Figure 2 shows a schematic diagram of a fragment analysis method using capillary electrophoresis according to the present invention. A primer 200 is used, which includes a double-stranded nucleic acid tag 204 with an interstrand crosslink 203 and a primer portion 205 that specifically binds to a target nucleic acid. A single-base extension reaction is performed using the target nucleic acid 201 as a template, and the product, to which a modified molecule 202 has been attached at the end, is subjected to capillary electrophoresis (CE) to perform genetic analysis of the target nucleic acid. By varying the length of the interstrand crosslinked double-stranded nucleic acid tag 204, the primer can be labeled in a manner that allows it to be distinguished by differences in mobility in CE. Furthermore, because the tag 204 is an interstrand-crosslinked double-stranded nucleic acid, nonspecific binding during the single-base extension reaction can be prevented, eliminating the difficulties in chemical synthesis and non-target binding that occur when using single-stranded nucleic acids, as well as the non-target binding that occurs after dissociation when using non-crosslinked double-stranded nucleic acids.

[0019] Thus, in one aspect, the present invention relates to a primer comprising a double-stranded nucleic acid tag having an interstrand bridge and a primer nucleic acid that specifically binds to a target nucleic acid.

[0020] The double-stranded nucleic acid tag having an interstrand bridge may be any of DNA, RNA, or hybrid nucleic acid, as long as it is a nucleic acid having a double-stranded nucleic acid structure. Preferably, the double-stranded nucleic acid is double-stranded DNA.

[0021] The double-stranded nucleic acid tag has at least one interstrand crosslink. In the present invention, "interstrand crosslink" means that one strand of a double-stranded nucleic acid is crosslinked to the other strand at at least one location. The method for intramolecularly crosslinking the two strands is not particularly limited as long as it is a method known in the art. Preferably, the interstrand crosslinking is achieved by photocrosslinking.

[0022] Interstrand crosslinking can be achieved using classically known crosslinking molecules such as nitrogen mustard, cisplatin, carmustine, mitomycin C, psoralen, trioxane (trimethylpsoralen), and malondialdehyde (see, for example, Guainazzi et al., Cellular and Molecular Life Sciences, 67:3683-3697, 2010). These crosslinking molecules insert one molecule between bases, either between AT or GC residues. Therefore, the crosslinking position is random across the entire nucleic acid molecule, and the crosslinking efficiency is approximately 30-40%. For example, psoralen is a photocrosslinking agent that generates photocrosslinks at 5'-TA-3' sequences upon photoreaction at a photoligation wavelength of 350 nm and cleaves the crosslink at a photocleavage wavelength of 250 nm.

[0023] Furthermore, for inter-chain crosslinking, 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) and 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), which are known as crosslinking molecules that can be introduced into oligo backbones, can be used (for example, Patent No. 4940311, Yoshimura et al., ChemBioChem, 10:1473-1476, 2009, Sakamoto et al., Org. Lett., 17:936-939, 2015). These molecules can form crosslinks by [2+2] cyclization with a pyrimidine base (thymine, cytosine, or uracil) in the complementary strand, one base away, triggered by irradiation with ultraviolet light (366 nm). Furthermore, because they can be incorporated into the nucleic acid molecular backbone, the crosslinks can be arbitrarily designed, making them practically advantageous. Another important practical feature of these photocrosslinkers is that they can reversibly dissociate the interstrand crosslinks upon excitation with ultraviolet light of a different wavelength (312 nm). The photocrosslinkers CNV-K and CNV-D are particularly suitable in terms of availability, cost, and other factors.

[0024] Other crosslinking molecules may be those formed using the Click reaction, which combines an azide group (-N3) with an alkyne group. Such crosslinking sites have been reported, for example, by Kocalta et al., ChemBioChem, 9:1280-1285, 2008. Examples of crosslinking molecules specific to specific base sequences include UTA-6026, which is specific to the sequence 5'-CAATTA-3' / 3'-GTTAAT-5' and bridges the AG residues separated by five bases (Zhou et al., J. Am. Chem. Soc., 123:4865-4866, 2001). Other known cross-linking molecules include ImImPy (Bando et al., J. Am. Chem. Soc., 123:5158-5159, 2001), which is specific for the sequence 5'-Py(T / C)GGC(T / A)GCCPu(A / G)-3' and bridges bases 9 bases apart, and C8 / C8'-tripyrrole-linked sequence-selective pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer (Tiberghien et al., Bioorganic & Medicinal Chemistry Letters, 18:2073-2077, 2008), which is specific for the sequence 5'-GCTTATAATGG-3' and bridges bases 11 bases apart. The advantage of these sequence-specific cross-linking molecules is that the cross-linking site can be designed.

[0025] A double-stranded nucleic acid tag having an interstrand crosslink determines the migration distance (mobility) in electrophoresis. That is, by linking double-stranded nucleic acid tags of different lengths to a primer, the migration distance in electrophoresis can be changed. Capillary electrophoresis can detect nucleic acids with chain lengths up to approximately 600 bases. Therefore, excluding the chain length (10 to 30 bases) of the primer nucleic acid that specifically binds to the target nucleic acid, the length of the double-stranded nucleic acid tag can range from 1 to approximately 590 bases. Furthermore, the base length of a double-stranded nucleic acid tag that results in a distinguishable migration distance is 1 base. For example, double-stranded nucleic acid tags that differ in length by 5 bases or more, preferably 10 bases or more, can be used in combination.

[0026] The base sequence of the double-stranded nucleic acid tag is not particularly limited as long as it is a nucleic acid having an interstrand crosslink. The double-stranded nucleic acid tag can be chemically synthesized by a known oligonucleotide synthesis method, but is usually synthesized using a commercially available chemical synthesis device.

[0027] The primer nucleic acid that specifically binds to the target nucleic acid (also referred to as a selective primer in this specification) may be either DNA or RNA, and is determined depending on the type of target nucleic acid and the type of polymerase used in the single-base extension reaction. Preferably, the primer nucleic acid is DNA, and the single-base extension reaction is carried out using DNA or mRNA as the target nucleic acid template.

[0028] Primer nucleic acids are designed to have a sequence that specifically binds to a target nucleic acid (or target region), i.e., a sequence complementary to the target nucleic acid (or target region). Primer design techniques are well known in the art, and primers usable in the present invention are designed to satisfy conditions that enable specific annealing, for example, to have a length and base composition (melting temperature) that enable specific annealing. For example, the length that functions as a primer is preferably 10 bases or more, more preferably 15 to 50 bases, and even more preferably 15 to 30 bases, e.g., approximately 20 bases. Furthermore, during design, it is preferable to confirm the GC content and melting temperature (Tm) of the primer. Tm refers to the temperature at which 50% of any nucleic acid strand forms a hybrid with its complementary strand. Optimizing the annealing temperature is necessary to form a double-stranded annealing between the template target nucleic acid and the primer. On the other hand, lowering this temperature too much can lead to nonspecific reactions, so it is desirable to keep the temperature as high as possible. Known primer design software can be used to confirm the Tm. The designed primers can be chemically synthesized by known oligonucleotide synthesis techniques, but are usually synthesized using a commercially available chemical synthesizer.

[0029] The primer of the present invention comprises an interstrand bridge double-stranded nucleic acid tag and a selective primer, which can be linked by any method. For example, a primer of the present invention can be prepared by preparing a sequence in which one strand of a double-stranded nucleic acid tag is linked to a selective primer directly or via a spacer, and then annealing the other strand of the double-stranded nucleic acid tag to form an interstrand bridge at at least one site in the double-stranded nucleic acid portion (e.g., Figure 3). Alternatively, a double-stranded nucleic acid tag having an interstrand bridge can be prepared and then linked to the selective primer directly or via a spacer (e.g., Figure 5). The linking method can be hydrogen bonding based on the complementarity of the base sequences, or ligation can be performed using a known ligase.

[0030] To easily prepare double-stranded nucleic acid tags of different lengths, for example, double-stranded nucleic acid tag units having overhanging ends as shown in Figure 7A can be linked in tandem as shown in Figure 7B. By changing the number of units linked in tandem, tags of different lengths can be easily prepared. Furthermore, by using such double-stranded nucleic acid tag units, primers can be easily tagged by simply binding (labeling) the double-stranded nucleic acid tag to the selective primer. In particular, by binding (labeling) double-stranded nucleic acid tags of different lengths to different selective primers, it becomes possible to prepare multiple primer sets that can be distinguished by their difference in length (i.e., difference in migration distance).

[0031] Therefore, in another aspect, the present invention provides a primer labeling kit comprising an interstrand-bridged double-stranded nucleic acid molecule, the interstrand bridged duplex nucleic acid molecule comprises at least one interstrand bridged duplex nucleic acid unit; the interstrand bridged double-stranded nucleic acid unit is a first oligonucleotide comprising a first base sequence comprising at least one interstrand-bridge-forming base and a second base sequence comprising at least one interstrand-bridge-forming base; a second oligonucleotide comprising a sequence that is complementary to the second base sequence and includes a base that forms an interstrand bridge with the interstrand bridge-forming base in the second base sequence, and a sequence that is complementary to the first base sequence and includes a base that forms a bridge with the interstrand bridge-forming base in the first base sequence; wherein the first base sequence in the first oligonucleotide and the sequence complementary to the first base sequence in the second oligonucleotide form a double-stranded nucleic acid.

[0032] The interstrand bridged double-stranded nucleic acid molecule is a first oligonucleotide comprising a first base sequence comprising at least one interstrand-bridge-forming base and a second base sequence comprising at least one interstrand-bridge-forming base; a second oligonucleotide comprising a sequence that is complementary to the second base sequence and includes a base that forms an interstrand bridge with the interstrand bridge-forming base in the second base sequence, and a sequence that is complementary to the first base sequence and includes a base that forms a bridge with the interstrand bridge-forming base in the first base sequence; and at least one interstrand bridged double-stranded nucleic acid unit comprising:

[0033] The first oligonucleotide may contain other sequences (e.g., spacer sequences) as long as it contains the first and second base sequences. Similarly, the second oligonucleotide may contain other sequences (e.g., spacer sequences) as long as it contains a sequence complementary to the second base sequence and a sequence complementary to the first base sequence.

[0034] The interstrand cross-linking base is preferably a photoresponsive interstrand cross-linking base as described above. For example, the pair of the interstrand cross-linking base and the base that forms an interstrand cross-link with the interstrand cross-linking base can be a pair of CNV-K or CNV-D and a pyrimidine base (thymine, cytosine, or uracil) that forms a cross-linking point by undergoing a [2+2] cyclization reaction with a pyrimidine base (thymine, cytosine, or uracil) that is located one base apart in a complementary strand. In this specification, when the interstrand cross-linking base is CNV-K or CNV-D, the base that forms the interstrand cross-link with the interstrand cross-linking base is a pyrimidine base, and when the interstrand cross-linking base is a pyrimidine base, the base that forms the interstrand cross-link with the interstrand cross-linking base is CNV-K or CNV-D.

[0035] An interstrand bridged double-stranded nucleic acid unit is formed by a double-stranded nucleic acid consisting of a first base sequence in a first oligonucleotide and a sequence complementary to the first base sequence in a second oligonucleotide. For example, in Figure 7A, if the sequence shown at the top (Core01-Lower01) is the first oligonucleotide and the 10 bases on the 5' side are the first base sequence, the two sequences form a double-stranded nucleic acid, with the 10 bases on the 5' side of the sequence shown at the bottom (Upper01-Core01-2) being complementary to the first base sequence in the second oligonucleotide. With such a double-stranded nucleic acid as one unit, an interstrand bridged double-stranded nucleic acid molecule contains at least one interstrand bridged double-stranded nucleic acid unit.

[0036] In one embodiment, the interstrand bridged duplex nucleic acid molecule contains two or more interstrand bridged duplex nucleic acid units. In this case, the two or more interstrand bridged duplex nucleic acid units are linked by forming a duplex nucleic acid between a second base sequence in a first oligonucleotide and a sequence complementary to the second base sequence in a second oligonucleotide. For example, in Figure 7B, an interstrand bridged duplex nucleic acid molecule containing multiple units can be prepared by tandemly linking units such as those shown in Figure 7A.

[0037] In one embodiment, the primer labeling kit includes a plurality of interstrand bridged duplex nucleic acid molecules containing different numbers of interstrand bridged duplex nucleic acid units, allowing different selective primers to be distinguishably and conveniently labeled with interstrand bridged duplex nucleic acid molecules of different lengths (containing different numbers of units).

[0038] The primer labeling kit may contain, in addition to the interstrand-bridged duplex nucleic acid molecule, other components used in primer labeling (such as a buffer and, if necessary, a ligase), instructions, and the like.

[0039] The primer of the present invention (a primer comprising a double-stranded nucleic acid tag having an interstrand crosslink and a selective primer) can be used, for example, in genetic analysis, specifically, detection of a target nucleic acid, determination of the base of the target nucleic acid, etc. Genetic analysis can be performed by any method that can distinguish test subjects based on differences in length. For example, capillary electrophoresis (CE), genetic analysis by electrophoresis, can be used.

[0040] Therefore, in a further aspect, the present invention provides a genetic analysis kit comprising a primer comprising a double-stranded nucleic acid tag having an interstrand bridge and a primer nucleic acid that specifically binds to a target nucleic acid. The genetic analysis kit comprises at least one primer. In a preferred embodiment, the genetic analysis kit comprises a plurality of primers comprising double-stranded nucleic acid tags of different lengths and primer nucleic acids that specifically bind to different target nucleic acids.

[0041] In addition to the primers, the gene analysis kit may also contain a buffer constituting a reaction solution, a dNTP or ddNTP mixture (which may be labeled), enzymes (polymerase, reverse transcriptase, etc.), a standard sample for calibration, etc. Providing the primers of the present invention as a kit enables gene analysis to be performed more quickly and easily.

[0042] In another aspect, the present invention provides a method for detecting a target nucleic acid in a sample and / or determining the base of a target nucleic acid, the method comprising: providing a sample containing or suspected of containing a target nucleic acid; providing 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; The resulting reaction product is subjected to capillary electrophoresis for analysis. This includes:

[0043] 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 acid, and any sample, including biologically derived samples (e.g., cell samples, tissue samples, liquid samples, etc.) and synthetic samples (e.g., nucleic acid libraries such as cDNA libraries, etc.), can be used. In the case of biologically derived samples, the organism from which the sample is derived is also not particularly limited, and samples derived from any organism, such as vertebrates (e.g., mammals, birds, reptiles, fish, amphibians, etc.), invertebrates (e.g., insects, nematodes, crustaceans, etc.), protists, plants, fungi, bacteria, and viruses, can be used. For example, when cancer testing in humans is envisioned, a nucleic acid-containing sample, such as whole blood, serum, plasma, saliva, urine, feces, skin tissue, or cancer tissue, obtained from the human subject is prepared.

[0044] The target nucleic acid is not particularly limited as long as it contains the sequence or base to be detected or determined, and includes deoxyribonucleic acid (DNA), such as genomic DNA and cDNA, and ribonucleic acid (RNA), such as messenger RNA (mRNA), and fragments thereof. In the present invention, cell-free DNA (cfDNA, DNA free in blood) and circulating tumor DNA (ctDNA) are preferably used as the target nucleic acid. Nucleic acids can be prepared from samples by methods known in the art. For example, when preparing target nucleic acids from blood or cells, cells can be lysed using protease 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, and the nucleic acids contained therein, i.e., DNA and RNA, can be eluted. When preparing RNA, DNA from the nucleic acids eluted by the above cell lysis is degraded with DNase, resulting in a sample containing only RNA as nucleic acid. When preparing mRNA, because mRNA contains a poly(A) sequence, it is possible to capture only mRNA from the RNA sample prepared as described above using a DNA probe containing a poly(T) sequence. Kits for preparing such nucleic acids are commercially available from many manufacturers, making it possible to easily purify the target nucleic acid.

[0045] Also, a primer is prepared, which includes a double-stranded nucleic acid tag having an interstrand bridge and a primer nucleic acid that specifically binds to a target nucleic acid. As described above, the double-stranded nucleic acid tag has a length that can be distinguished by mobility, and the primer nucleic acid is designed to specifically bind to the target nucleic acid and cause a single-base extension reaction.

[0046] In one embodiment, this method uses multiple primers containing double-stranded nucleic acid tags of different lengths and primer nucleic acids that specifically bind to different target nucleic acids. As described above, the base length of the double-stranded nucleic acid tag that results in a distinguishable migration distance is approximately 15 to 20 bases, so double-stranded nucleic acid tags that differ in length, for example, by 15 or more bases, preferably 20 or more bases, are bound to different primers. This method makes it possible to simultaneously detect, for example, one to about 100 different target nucleic acids.

[0047] Subsequently, a single-base extension reaction is carried out using a primer with the target nucleic acid as a template. Single-base extension reactions are known in the art and are typically performed using a polymerase. The polymerase used is selected depending on the type of template (target nucleic acid) and the type of primer 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.

[0048] The single-base extension reaction is widely known in the art, and for example, Non-Patent Document 3 describes a method for efficiently extending one base by a cycle reaction.

[0049] When a target nucleic acid is present, a selective primer that specifically binds to this target nucleic acid hybridizes, and a base is incorporated as a substrate from the 3'-end portion of the selective primer by the synthesis reaction of the polymerase. At this time, by using, for example, a dideoxynucleotide (ddNTP) as the base (substrate) to be incorporated, the synthesis reaction is completed with only one base extension. In one embodiment, a modified base, for example, a labeled ddNTP, is used as the substrate to carry out the one-base extension reaction. The label is useful for easily detecting whether or not the base has been incorporated, or for determining the type of the incorporated base, and labels known in the art can be used. Such labels include radioisotopes ( 32 P, 125 I, 35Examples include fluorescent substances and luminescent substances (such as luciferin). Fluorescent substances can be preferably used, and examples thereof include, but are 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 (registered trademark), ATTO series, Alexa Fluor (registered trademark) series, Cy series, and the like. Fluorescent dyes that do not cause a shift in migration size include dR110 (carboxy-dichloro rhodamine Examples of such labels include 110), dR6G (dihydro rhodamine 6G), dTAMRA (tetramethyl rhodamine), and dROX (carboxy-X-rhodamine). For example, when attempting to determine the type of base, five fluorescent substances that are excited and detected at different wavelengths can be used in combination to distinguish between four types of bases and five types for reference (to detect and correct base length from a reference ladder DNA). The type of label and the method for introducing the label are not particularly limited, and various conventionally known means can be used. In a preferred embodiment, fluorescently labeled dideoxynucleotides (ddNTPs) are used as modified bases.

[0050] The presence or absence of a target nucleic acid can be determined by whether or not this one-base extension occurs, and the specific base in the target nucleic acid can be determined based on the type of base incorporated into the one-base extension portion. 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, the selective primer is hybridized to the target nucleic acid, and a one-base extension reaction is performed using a base with a different label as a substrate. By determining the type of the incorporated base based on the label, the SNP in the target nucleic acid can be detected.

[0051] After the single-base extension reaction, the resulting reaction product is subjected to capillary electrophoresis (CE) for analysis. CE is a technique for separating introduced components based on differences in mobility, which are determined by factors such as charge, size, and shape. This method utilizes double-stranded nucleic acid tags that cause differences in mobility. Based on the mobility, the type of target nucleic acid (based on the double-stranded nucleic acid tag bound to the selective primer) and the presence or absence of the target nucleic acid or the type of specific base in the target nucleic acid (based on the single-base extension reaction) can be used to detect the target nucleic acid and / or determine the base of the target nucleic acid.

[0052] The present invention should not be construed as being limited to the description of the following examples. Those skilled in the art will readily understand that the specific configurations can be modified without departing from the spirit or scope of the present invention.

[0053] In order to facilitate understanding of the invention, the position, size, shape, range, etc. of each component shown in the drawings etc. may not represent the actual position, size, shape, range, etc. Therefore, the present invention is not necessarily limited to the position, size, shape, range, etc. disclosed in the drawings etc.

[0054] The publications and patent documents cited in this specification are incorporated herein by reference in their entirety. As used herein, elements referred to in the singular are intended to include the plural unless the context clearly indicates otherwise.

[0055] [Example 1] In this example, selective primers containing interstrand cross-linked double-stranded DNA tags as labels were designed and their mobility in acrylamide gel electrophoresis was examined. The designed base sequence for the interstrand cross-linking test is shown in Figure 3. In this example, the interstrand cross-linking oligo special base (CNV-D: 3-O-(4,4'-Dimethoxytrityl)-2-N-(N-carboxy-3-cyanovinylcarbazol)-D-threonin-1-yl-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) that forms an interstrand cross-link upon UV irradiation, as disclosed in Japanese Patent No. 4940311, was used.

[0056] As shown in Figure 3, two complementary oligo DNA molecules, CNV02 (19-mer: SEQ ID NO: 1) and RC_CNV02 (47-mer: SEQ ID NO: 2), were designed. The N base (boxed in Figure 3) in one of the oligo DNA molecules (SEQ ID NO: 1) is a special base that forms a photocrosslink, and three photocrosslinking oligos were inserted into the short DNA molecule (19-mer: SEQ ID NO: 1). The N base (CNV-D) in CNV02 crosslinks with the pyrimidine base (C or T base; bold and underlined in Figure 3) one base upstream of the complementary strand, RC_CNV02 (SEQ ID NO: 2), upon 366 nm UV irradiation.

[0057] The crosslinking reaction was carried out using a 365 nm UV irradiation device (ULEDN-102CT, NS Lighting Co., Ltd.) with irradiation conditions set at 62 mW and 1 second. The solution composition used during the crosslinking reaction was the standard 1x concentration of KOD buffer included with KOD Polymerase (TOYOBO), a PCR enzyme.

[0058] Figure 4 shows acrylamide gel electrophoresis images of the crosslinking reaction products. As indicated by the arrows in Figure 4, in lane 1, where uncrosslinked RC_CNV02 (47mer: SEQ ID NO: 2) was electrophoresed, DNA fragments shorter than 50 bp were observed, whereas in lane 2, where the UV-irradiated crosslinked product was electrophoresed, DNA fragments longer than 50 bp were observed. These experimental results demonstrated that UV irradiation causes interstrand crosslinks to form, that the electrophoretic mobility changes due to the formation of interstrand crosslinks, and that DNA after photocrosslinking is detected at a base position longer than the chain length.

[0059] [Example 2] In this example, we verified the fluorescently labeled single-base extension reaction using a primer linked to an interstrand cross-linked double-stranded DNA tag as a label. The target gene was the EGFR gene, a gene mutation frequently found in colorectal and lung cancers, and primer DNA was created that linked a primer region specific to the gene mutation that appears at position 788 (Figure 5).

[0060] The primers consisted of three oligonucleotides: the Lower01 oligo (20-mer, SEQ ID NO: 3) photocrosslinked to the upstream Core01-Lower01 oligo (20-mer, SEQ ID NO: 4) and the downstream EGFR L858-Lower-FW1 oligo (38-mer, SEQ ID NO: 5). The 20-base single-stranded DNA portion from the 3' end of the EGFR L858-Lower-FW1 oligo specifically recognized the EGFR gene (double underlined portion in Figure 5). In this experiment, the EGFR gene was selected as the target gene, but the complementary sequence to Lower01 allowed any target-specific primer to be linked to Lower01. As shown in Figure 5, the labeled primer may contain any single-stranded DNA sequence or spacer region, as long as it contains the interstrand-bridged double-stranded DNA tag portion and the selective primer DNA portion.

[0061] Fluorescent single-base extension reactions using the EGFR gene sequence as the target template were performed using a mixture of 1 μL of 10x Therminator buffer (NEB), 0.5 μL of Therminator (NEB), 1 μL of ddNTPs (10 μM), 1 μL of template DNA (100 pmol / μL), 1 μL of the above primers, and 5.5 μL of DW. The reaction was performed using a thermal cycler (96°C, 10 sec), followed by 40 cycles of (50°C, 5 sec), followed by (60°C, 30 sec). The reaction solution was purified with alkaline phosphatase (TAKARA) and then analyzed using a CE sequencer, SeqStudio (Thermo Fisher Scientific).

[0062] The results of fragment analysis using a CE sequencer are shown in Figure 6. When the unlabeled EGFR L858-Lower-FW1 primer (SEQ ID NO: 5) was used, a fluorescent signal was detected only around 40 bp, corresponding to a 38-mer primer length (Figure 6A). With the labeled primer, multiple fluorescent signals were observed in the 70-80 bp range in addition to around 40 bp (Figure 6B). In this experiment, the product of the cross-linking treatment reaction was used as a primer for the fluorescently labeled single-base extension reaction; therefore, the signal at 40 bp observed in Figure 6B is a signal derived from the remaining unlabeled EGFR L858-Lower-FW1. The signal in the 70-80 bp range observed in Figure 6B is a fluorescent signal whose migration position has shifted due to labeling with the interstrand-cross-linked double-stranded DNA tag.

[0063] Furthermore, these results confirmed that the interstrand cross-linked structure withstood 40 cycles of thermal dissociation treatment used in the fluorescently labeled single-base extension reaction and maintained the double strand. Since the double strand was maintained during the thermal treatment cycle, it was demonstrated that the interstrand cross-linked double-stranded DNA of the present invention has a structure that does not bind to other primers and does not undergo nonspecific binding during the fluorescent single-base extension reaction.

[0064] [Example 3] In this example, we verified the creation of interstrand-crosslinked double-stranded nucleic acid tags that form a tandem structure, with the aim of creating nucleic acid tags with diverse mobilities. The structure of the designed double-stranded nucleic acid tag (1 unit) is shown in Figure 7A.

[0065] The interstrand-crosslinked double-stranded DNA tag unit shown in Figure 7A has a base sequence at the protruding end that allows for the formation of a tandem structure during double-strand formation, and as shown in Figure 7B, it is possible to synthesize interstrand-crosslinked double-stranded DNA molecules of various lengths depending on the number of ligations during UV crosslinking. This makes it possible to easily prepare sets of labeled tags of different lengths.

[0066] Crosslinking was performed under the same conditions as in Example 1, and the results of evaluation by acrylamide electrophoresis are shown in Figure 8. The electrophoretic image also confirmed the presence of ladder-like DNA fragments consisting of approximately 20 bp base units. These experimental results demonstrated that double-stranded nucleic acid labeling tags of various lengths can be synthesized by designing the unit structure that forms the tandem structure, and that evenly spaced mobility-shifting tags can be produced by linking the same structural units. [Explanation of symbols]

[0067] 100...Selective primer 101, 201...Template molecules 102, 202…modified molecule 200...Primer 203…Interchain crosslinking 204...Double-stranded nucleic acid tag with interstrand bridge 205...Selective primer nucleic acid [Sequence List Free Text]

[0068] SEQ ID NOs: 1 to 7: Artificial (synthetic oligonucleotides)

Claims

1. 1. A method for detecting the presence of a target nucleic acid in a sample and / or determining the base of a target nucleic acid, the sample containing or suspected of containing a target nucleic acid, comprising: providing 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 in the sample as a template and a fluorescently labeled modified base as a substrate, and the primer; The resulting reaction product is subjected to capillary electrophoresis for analysis. The method includes:

2. The method of claim 1 , wherein the primers comprise a plurality of primers each comprising a double-stranded nucleic acid tag of a different length and a primer nucleic acid that specifically binds to a different target nucleic acid.

3. 2. The method of claim 1, wherein the modified base comprises a fluorescently labeled dideoxynucleotide (ddNTP).

4. A gene analysis kit for use in the method of claim 1, comprising a primer characterized by comprising a double-stranded nucleic acid tag having an interstrand bridge and a primer nucleic acid that specifically binds to a target nucleic acid.

5. The kit of claim 4 , wherein the double-stranded nucleic acid tag defines a migration distance in electrophoresis.

6. The kit of claim 4 , wherein the double-stranded nucleic acid tag has at least one interstrand bridge.

7. The kit of claim 4 , wherein the inter-strand crosslinking is by photocrosslinking.

8. The kit of claim 4 , wherein the double-stranded nucleic acid is double-stranded DNA.

9. The kit of claim 4 , wherein the primers comprise a plurality of primers each comprising a double-stranded nucleic acid tag of a different length and a primer nucleic acid that specifically binds to a different target nucleic acid.

10. A primer labeling kit comprising an interstrand-bridged double-stranded nucleic acid molecule, the interstrand bridged duplex nucleic acid molecule comprises at least one interstrand bridged duplex nucleic acid unit; the interstrand bridged double-stranded nucleic acid unit is a first oligonucleotide comprising a first base sequence comprising at least one interstrand-bridge-forming base and a second base sequence comprising at least one interstrand-bridge-forming base; a second oligonucleotide comprising a sequence that is complementary to the second base sequence and includes a base that forms an interstrand bridge with the interstrand bridge-forming base in the second base sequence, and a sequence that is complementary to the first base sequence and includes a base that forms a bridge with the interstrand bridge-forming base in the first base sequence; wherein a first base sequence in the first oligonucleotide and a sequence complementary to the first base sequence in the second oligonucleotide form a double-stranded nucleic acid.

11. The kit according to claim 10 , wherein the interstrand cross-linking base is a photoresponsive interstrand cross-linking base.

12. The kit of claim 10, wherein the interstrand bridged double-stranded nucleic acid molecule comprises two or more interstrand bridged double-stranded nucleic acid units, and the two or more interstrand bridged double-stranded nucleic acid units are linked by forming a double-stranded nucleic acid between a second base sequence in a first oligonucleotide and a sequence complementary to the second base sequence in a second oligonucleotide.

13. The kit of claim 10, comprising a plurality of interstrand-bridged duplex nucleic acid molecules comprising different numbers of said interstrand-bridged duplex nucleic acid units.

Images