Long chain ssdna primer for base detection

Abstract

To provide a method and means for synthesizing long single-stranded (ss) DNA primers for use in a fluorescent labeling base extension reaction method to realize simultaneous detection of multiple target nucleic acids by expanding the analytical range of a capillary electrophoresis (CE) sequencer.SOLUTION: Provided is a method for synthesizing a long single-stranded DNA (ssDNA) primer of 100 to 700 bases in length, comprising the steps of: using a forward primer to which a strand length determining sequence that determines the length of the ssDNA primer strand has been added and a reverse primer to which a sequence complementary to a target recognition sequence has been added at the 5' end, and amplifying a region in a template that contains a non-target sequence that is non-specific to a target nucleic acid, thereby preparing a double-stranded DNA fragment that contains the strand length determining sequence, the non-target sequence, and the target recognition sequence; and recovering ssDNA having the target recognition sequence at the 3' end from the double-stranded DNA fragment.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a single-stranded DNA (ssDNA) primer having a length of more than 200 mer and a method for synthesizing the same, and also to a gene analysis method using the ssDNA primer. [Background technology]

[0002] There are many individual differences in genomes, and the differences in genome sequences are useful biomarkers for disease and drug response. Therefore, there is a need for low-cost detection of genome sequence mutations in diagnostic and clinical applications.

[0003] The main methods used to detect genome mutations are detection by PCR, base sequence analysis using a sequencer, and single-base extension reaction analysis (Patent Document 1).

[0004] Since the fluorescent labeling single-base extension reaction method uses electrophoretic mobility as an index for gene identification, it is possible to simultaneously detect multiple mutations by adjusting the length of the primers used or by attaching a label that changes the mobility to the primers, thereby changing the position at which the signal is detected. It is also possible to detect the gene or mutation to be detected by extending multiple bases rather than extending one base, depending on the gene or mutation to be detected. In Non-Patent Document 1, simultaneous detection of 26 plex (26 types of mutations) is performed, but the range used for analysis is a region of 120 bp within the analysis range of a capillary electrophoresis (CE) sequencer. A CE sequencer has the accuracy to separate a chain length difference of one base within a range of 50 to 600 bp (up to 700 bp depending on the device), but the existing fluorescent labeling single-base extension reaction method only uses a part of the detection chain length range of the CE sequencer.

[0005] One of the reasons why the detection chain length range of CE sequencers is limited is that it is difficult to synthesize >200 bp in the chemical synthesis of oligo DNA used as a primer. To solve this problem, mobility correction using labels other than nucleic acids, as in Meaghe et al. (Non-Patent Document 2), is also effective, but it is not easy to synthesize many types of these polymers with high purity and polymerization degree.

[0006] In addition, the PCR product used as a template in the base measurement by the fluorescent label single base extension reaction method was found to be a source of noise signals, and the kit used in this method, ABI PRISM (registered trademark) SNaPshot TM This is described in the troubleshooting section of the protocol that comes with the Multiplex Kit. In general, the amplification size of multiplex PCR is considered to be approximately 200 bp (Non-Patent Document 3). In particular, the chain length of cell-free DNA contained in liquid biopsy, which is an important target sample for future genetic testing, is approximately 167 bp (Non-Patent Document 4), and PCR amplification products using liquid biopsy as a sample will have a fragment length of approximately 200 bp. In the fluorescent labeling single base extension reaction method, the vicinity of the limit length of primer chemical synthesis overlaps with the noise signal region derived from this PCR amplification product, making it difficult to use. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 5,888,819 [Non-patent literature]

[0008] [Non-Patent Document 1] Coutinho1 et, al., PLOS ONE, 9(3):e923292, 2014 [Non-Patent Document 2] Meagher et, al., Anal. Chem., 79(5):1848-1854, 2007 [Non-Patent Document 3] Thornton B. et al., Biochem Mol Biol Educ. 39(2):145-154, 2011 [Non-Patent Document 4] Lapin, M. et al., J. Transl. Med. 16(1):300, 2018 Summary of the Invention [Problem to be solved by the invention]

[0009] In order to improve the number of multiplexes (the number of items that can be detected simultaneously) in base measurement using the fluorescent-labeled single-base extension reaction method, it is necessary to synthesize primers that are longer than the 200-mer limit imposed by chemical synthesis and the target length of the template PCR product, 200 bp. [Means for solving the problem]

[0010] As a result of intensive development into the synthesis of long-chain ssDNA primers with the aim of diversifying the existing fluorescently labeled single-base extension reaction methods (and even fluorescently labeled base extension reaction methods not limited to single-base extension reactions), the inventors discovered a method for synthesizing long-chain ssDNA primers with a chain length of >200 mer and the usefulness of their base detection ability, which led to the completion of the present invention.

[0011] In one aspect, the present invention provides a method for synthesizing a long single-stranded DNA (ssDNA) primer having a length of 100 to 700 bases, comprising: a step of amplifying a region of a template containing a non-target sequence that is non-specific to the target nucleic acid, using a forward primer to which a length determination sequence that determines the length of the ssDNA primer has been added and a reverse primer to which a sequence complementary to a target recognition sequence has been added at the 5' end, thereby preparing a double-stranded DNA fragment containing the length determination sequence, the non-target sequence, and the target recognition sequence; a step of recovering ssDNA having the target recognition sequence at the 3' end from the double-stranded DNA fragment. The present invention relates to a method comprising the steps of:

[0012] In another aspect, the present invention relates to a reagent for use in genetic analysis by base extension reaction and electrophoresis, comprising the long ssDNA primer synthesized by the above method.

[0013] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: performing a base extension reaction on a test nucleic acid using the long ssDNA primer synthesized by the above method and a substrate for base extension reaction having a fluorescent dye; subjecting the reaction product of the base extension reaction to electrophoresis; detecting a target nucleic acid based on said electrophoretic mobility and said fluorescent dye. The present invention relates to a method for gene analysis comprising the steps of:

[0014] In yet another aspect, the present invention provides a kit for use in a method for synthesizing a long single-stranded DNA (ssDNA) primer having a length of 100 to 700 bases, comprising: a forward primer to which a length determination sequence that determines the length of the ssDNA primer has been added; and A template containing a non-target sequence that is non-specific to the target nucleic acid Including, The method relates to a kit comprising: preparing a double-stranded DNA fragment containing the chain length determination sequence, the non-target sequence, and the target recognition sequence by amplifying a region containing a non-target sequence in the template using the forward primer together with a reverse primer having a sequence complementary to the target recognition sequence added to the 5' end thereof; and recovering from the double-stranded DNA fragment an ssDNA having the target recognition sequence at its 3' end. Effect of the Invention

[0015] The use of long-stranded base extension primers for detecting target nucleic acids has enabled a wider range of detection strand lengths to be utilized in instruments used for fluorescent-labeled base extension reaction methods. This widens the detection strand length range, improving the number of multiplex detections in one run, i.e., the number of types of target nucleic acids that can be detected simultaneously. [Brief description of the drawings]

[0016] [Figure 1] 1 shows an outline of a method for synthesizing a long ssDNA primer. [Diagram 2] 1 shows an outline of a method for synthesizing a long ssDNA primer using a modified reverse primer. [Diagram 3] 1 shows an outline of a method for synthesizing a long ssDNA primer using a nuclease-resistant forward primer. [Figure 4] FIG. 2 is a schematic diagram showing the location of the phosphate-modified primers in the target nucleic acid used in the experiment of Example 1. [Diagram 5] 1 is a photograph showing an electrophoretic image of an amplified product obtained by PCR using the primers designed in Example 1. [Figure 6] 1 shows the results obtained by a fluorescently labeled single base extension reaction method (SNaPshot reaction) using a long ssDNA primer in Example 2. [Figure 7] 1 shows the results of analyzing a fluorescently labeled single base extension reaction method (SNaPshot reaction) using a long ssDNA primer in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention relates to a method and a kit for synthesizing a long-chain single-stranded DNA (ssDNA) primer, particularly a long-chain ssDNA primer for use in a fluorescently labeled base extension reaction method, as well as the obtained long-chain ssDNA primer and its use. The ssDNA primer has a sequence (target recognition sequence) at its 3' end that specifically binds to a sequence immediately preceding a base to be detected in a target nucleic acid, and as a result, after binding to the target nucleic acid, the primer is capable of incorporating a base corresponding to the base to be detected at the 3' end of the primer by a base extension reaction and extending it. The base length of the ssDNA primer affects the base length (size) of the reactant in the base extension reaction, and therefore the electrophoretic mobility is determined from the base length of the ssDNA primer. In the fluorescently labeled base extension reaction method, the base to be detected can be detected based on the type of the incorporated base and the electrophoretic mobility. Therefore, it is necessary to appropriately design the base sequence (target recognition sequence) and base length of the ssDNA primer.

[0018] Generally, the base length of the ssDNA primer is not limited as long as it has a target recognition sequence. In the present invention, a long-chain ssDNA primer with a base length that is considered difficult to chemically synthesize is synthesized, and the base length of the long-chain ssDNA primer is, for example, 100 bases or more, preferably 200 bases or more or more than 200 bases, more preferably 300 bases or more, 400 bases or more, or 500 bases or more. The upper limit of the base length of the ssDNA primer is the upper limit of the base length that can be analyzed in the device used for electrophoresis, and is, for example, 700 bases or less, but varies depending on the device used.

[0019] In one aspect, the present invention provides a method for synthesizing a long single-stranded DNA (ssDNA) primer having a length of 100 to 700 bases, comprising: a step of amplifying a region of a template containing a non-target sequence that is non-specific to the target nucleic acid, using a forward primer to which a length determination sequence that determines the length of the ssDNA primer has been added and a reverse primer to which a sequence complementary to a target recognition sequence has been added at the 5' end, thereby preparing a double-stranded DNA fragment containing the length determination sequence, the non-target sequence, and the target recognition sequence; a step of recovering ssDNA having the target recognition sequence at the 3' end from the double-stranded DNA fragment. The present invention provides a method comprising:

[0020] First, a region containing a non-target sequence in a template is amplified using a forward primer and a reverse primer, which are designed to contain a sequence (PCR recognition sequence) that anneals to the corresponding region of the template.

[0021] Design techniques for forward and reverse primers are well known in the art, and the forward and reverse primers usable in the present invention are designed to satisfy conditions for specific annealing, for example, to include a sequence (PCR recognition sequence) having a length and base composition (melting temperature) that allows 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, for example, about 20 bases. In addition, during design, it is preferable to confirm the GC content and melting temperature (Tm) of the primer. To confirm Tm, known primer design software can be used. The designed primer can be chemically synthesized by known oligonucleotide synthesis techniques, but is usually synthesized using a commercially available chemical synthesis device.

[0022] In addition to a sequence (PCR recognition sequence) that anneals to the template, a chain length determination sequence that determines the chain length of the ssDNA primer is added to the forward primer. As a result, when a region containing a non-target sequence is amplified, the chain length determination sequence is added to the double-stranded DNA fragment that is the amplified product. The specific sequence of the chain length determination sequence is not limited as long as it has a base length necessary to obtain an ssDNA primer of the desired base length. Therefore, the base length of the chain length determination sequence is determined based on the base lengths of the target recognition sequence and the non-target sequence and the base length of the desired ssDNA primer. For example, the chain length determination sequence can be 0 to 200 bases long, preferably 0 to 100 bases long. However, the chain length determination sequence does not include a sequence specific to the target. A person skilled in the art can appropriately design the specific sequence of the chain length determination sequence so that the chain length determination sequence does not form a hairpin structure.

[0023] In addition to a sequence (PCR recognition sequence) that anneals to the template, a sequence complementary to the target recognition sequence is added to the 5' end of the reverse primer. The target recognition sequence is a sequence specific to the target nucleic acid containing the base to be detected by the fluorescent labeling base extension reaction method, and specifically, is a sequence that specifically binds to the sequence immediately preceding the base to be detected in the target nucleic acid. In order to specifically bind to the target nucleic acid, the target recognition sequence is preferably 10 bases or more, more preferably 15 to 50 bases, and even more preferably 15 to 30 bases, for example, about 20 bases. The double-stranded DNA fragment amplified by this reverse primer contains a DNA strand having a target recognition sequence at the 3' end.

[0024] The template contains a non-target sequence and a sequence (PCR recognition sequence) to which the forward primer and reverse primer anneal. The base length and sequence of the non-target sequence are not limited as long as it does not contain a sequence specific to the target nucleic acid. For example, the non-target sequence may be 0 to 500 bases long, preferably 100 to 500 bases long. A person skilled in the art can appropriately design the specific sequence of the non-target sequence so that the non-target sequence does not form a hairpin structure in the ssDNA primer.

[0025] An amplification reaction is carried out using a forward primer, a reverse primer, and a template. The amplification reaction can be carried out by any amplification reaction known in the art. The enzyme used in the amplification reaction is a polymerase capable of carrying out an extension reaction using DNA as a template, and any known polymerase can be used. The conditions and reagents for the amplification reaction are well known in the art, and a person skilled in the art can select any of them.

[0026] By the amplification, a double-stranded DNA fragment containing a length determining sequence, a non-target sequence, and a target recognition sequence is prepared. From this double-stranded DNA fragment, ssDNA having a target recognition sequence at the 3' end is recovered. This makes it possible to synthesize an ssDNA primer of the desired base length (100 to 700 bases).

[0027] [Embodiment 1] The outline of the method for synthesizing long-chain ssDNA primers for use in the fluorescent-labeled base extension reaction method is shown in Figure 1. A set of primers for amplifying non-target sequences that do not have complementarity to the target nucleic acid, i.e., a forward primer 101 (Length control_1) (0-100 mer) that determines the length of the ssDNA primer (including the mobility design portion of the chain length determination sequence) and a reverse primer 102 (Target specific) (> 10 mer) that has a sequence complementary to the target recognition sequence at the 5' end, are used to amplify a DNA fragment 103 that contains a non-target sequence. A DNA strand that has the target recognition sequence at the 3' end is recovered from the double-stranded DNA fragment 104 obtained by amplification, thereby obtaining a long-chain ssDNA primer 105 for detecting the target base by the fluorescent-labeled base extension reaction method.

[0028] [Embodiment 2] FIG. 2 is a schematic diagram of a method for synthesizing a long-chain ssDNA primer, in which a reverse primer 202 is modified with biotin 206 in advance. A DNA fragment 203 containing a non-target sequence is amplified using a forward primer 201 (Length control_1) (0-100 mer) and a reverse primer 202 (Target specific) (> 10 mer) having a biotin modification 206 and a sequence complementary to a target recognition sequence at the 5' end. In the double-stranded DNA fragment 204 obtained by the amplification, the DNA strand having the target recognition sequence at the 5' end retains the biotin modification 206. The double-stranded DNA fragment 204 is adsorbed to streptavidin-modified beads, and by applying heat denaturation, the DNA strand 205 that is not adsorbed to the beads due to the lack of biotin modification is selectively recovered from the beads. This process produces a long-chain ssDNA primer 205 for detecting the target base by a fluorescent-labeled base extension reaction method.

[0029] Thus, in one embodiment, the method of the invention comprises: The reverse primer further has a modification at the 5' end, In the recovery step, ssDNA having a target recognition sequence at the 3' end is recovered from the double-stranded DNA fragment by utilizing the modification of the 5' end.

[0030] The modification of the 5' end of the reverse primer is not limited as long as it can distinguish and recover ssDNA in the recovery step, and examples of the modification include biotin modification, digoxigenin modification, amino group modification, and thiol modification. This makes it possible to simply and efficiently recover the ssDNA primer having a target recognition sequence at the 3' end by utilizing the 5' end modification.

[0031] [Embodiment 3] FIG. 3 is a schematic diagram of a method for synthesizing a long-chain ssDNA primer, in which a nuclease-resistant 307 is added to the 5' end of a forward primer 301. A DNA fragment 303 containing a non-target sequence is amplified using a forward primer 301 (Length control_1) having a nuclease-resistant 307 at its 5' end and a reverse primer 302 (Target specific) having a sequence complementary to a target recognition sequence at its 5' end. In a double-stranded DNA fragment 304 obtained by amplification, the 5' end of the DNA strand having a target recognition sequence at its 3' end retains nuclease resistance 307. By decomposing the double-stranded DNA fragment 304 with lambda exonuclease, which has DNA degradation activity from the 5' end to the 3' end, a DNA strand 305 whose 5' end retains nuclease resistance is selectively left. The DNA strand obtained by this process has a target recognition sequence at the 3' end and, due to the nuclease resistance at the 5' end, the ssDNA strand length is maintained even after treatment with DNase, making it a long-chain ssDNA primer suitable for detection of target bases by the fluorescent-labeled base extension reaction method.

[0032] Thus, in one embodiment, the method of the invention comprises: The forward primer further has a nuclease-resistant structure at the 5' end, In the recovery step, ssDNA having a target recognition sequence at the 3' end is recovered from the double-stranded DNA fragment using a nuclease having DNA degradation activity from the 5' end to the 3' end.

[0033] Nuclease resistance means resistance to 5'→3' degradation by nuclease. The nuclease-resistant structure of the forward primer is not particularly limited as long as it is a structure that is not degraded by a nuclease having DNA degradation activity from the 5' end to the 3' end. For example, when lambda exonuclease is used as such a nuclease, a dephosphorylated structure, a phosphorothioate bond, or the like can be used as the nuclease-resistant structure. This makes it possible to use the nuclease-resistant structure at the 5' end to degrade ssDNA that does not have a nuclease-resistant structure at the 5' end with a nuclease, and to simply and efficiently recover an ssDNA primer having a target recognition sequence at the 3' end.

[0034] The method of the present invention allows the synthesis of long ssDNA primers having a desired base length. By adjusting the base length according to the type of target recognition sequence, it is also possible to synthesize long ssDNA primers for each target recognition sequence, which can simultaneously detect multiple target recognition sequences by electrophoresis.

[0035] In another aspect, the present invention provides a long ssDNA primer synthesized by the method of the present invention.The present invention also provides a reagent for use in genetic analysis by base extension reaction and electrophoresis, comprising the long ssDNA primer synthesized by the method of the present invention.

[0036] As described above, the long-chain ssDNA primer of the present invention includes a chain length determination sequence, a non-target sequence, and a target recognition sequence at the 3' end, and can be used in a fluorescently labeled base extension reaction method (genetic analysis by base extension reaction and electrophoresis). In one embodiment, the long-chain ssDNA primer of the present invention has a nuclease-resistant structure at the 5' end. The long-chain ssDNA primer of the present invention has a length that is difficult to synthesize by chemical synthesis, and can maximize the use of 50 to 700 bases, which is the detection range of the equipment used in the fluorescently labeled base extension reaction method (e.g., a capillary electrophoresis device). Therefore, the long-chain ssDNA primer of the present invention makes it possible to simultaneously detect more target nucleic acids with high sensitivity in the fluorescently labeled base extension reaction method. Here, the fluorescently labeled base extension reaction method is preferably a fluorescently labeled single-base extension reaction method (genetic analysis by single-base extension reaction and electrophoresis).

[0037] In another aspect, the present invention provides a method for gene analysis using the long ssDNA primer synthesized by the method of the present invention. Specifically, the method for gene analysis of the present invention comprises the steps of: carrying out a base extension reaction on a test nucleic acid using a long ssDNA primer synthesized by the method of the present invention and a substrate having a fluorescent dye; subjecting the reaction product of the base extension reaction to electrophoresis; detecting a target nucleic acid based on said electrophoretic mobility and said fluorescent dye. Includes.

[0038] In one embodiment, the long-chain ssDNA primer comprises a plurality of long-chain ssDNA primers for a plurality of target nucleic acids. In this case, the plurality of long-chain ssDNA primers comprise target recognition sequences for the respective target nucleic acids and have different base lengths. In addition, in one embodiment, the long-chain ssDNA primer may be used in combination with a ssDNA primer (not necessarily long). By using a plurality of ssDNA primers with different base lengths, it is possible to simultaneously detect a plurality of target nucleic acids (in one run).

[0039] The genetic analysis method of the present invention is based on a fluorescently labeled base extension reaction method, i.e., a genetic analysis method that combines a base extension reaction and electrophoresis (preferably a fluorescently labeled single-base extension reaction method), and such genetic analysis methods are well known in the art, for example, as described in Non-Patent Documents 1 and 2 and Patent Document 1.

[0040] The base extension reaction is carried out using a base extension reaction primer (including a long-chain ssDNA primer in the present invention) for detecting a target nucleic acid in the presence of a substrate (dideoxynucleotide triphosphate: ddNTP) bound to a fluorescent dye.

[0041] In the present invention, the target nucleic acid refers to a nucleic acid containing a base sequence that is desired to be detected (and quantified). For example, the target nucleic acid contains a base sequence that contains a genetic mutation, and the target nucleic acid is detected (and quantified) by distinguishing between a wild type and a mutant type. Examples of such genetic mutations include single nucleotide polymorphisms (SNPs), insertion mutations, deletion mutations, and the like, but are not limited thereto. Furthermore, the mutant type of the target nucleic acid may include one type of mutant type or multiple types of mutant types. For example, there is a genetic mutation that has a wild type and two to three types of mutant types, and such multiple types of mutant types can also be detected (and quantified) by distinguishing them.

[0042] The test nucleic acid is not particularly limited as long as it is a sample to be detected for the target nucleic acid, and includes deoxyribonucleic acid (DNA), such as genomic DNA, cDNA, cell-free DNA (cfDNA, DNA free in blood), circulating tumor DNA (ctDNA), and ribonucleic acid (RNA), such as messenger RNA (mRNA), and fragments thereof. The preparation of nucleic acid can be carried out by a method known in the art.

[0043] In the genetic analysis method, a base extension reaction is carried out using the above-mentioned ssDNA primer in the presence of a substrate having a fluorescent dye. The base extension reaction is known in the art, and the polymerase used is selected depending on the type of template (test nucleic acid). For example, a DNA-dependent or RNA-dependent DNA polymerase is used for the base extension reaction using a DNA primer with DNA or RNA as a template, respectively.

[0044] When a target nucleic acid is present, the ssDNA primer hybridizes to the target nucleic acid, and a nucleotide is incorporated as a substrate from the 3'-end of the primer by a polymerase synthesis reaction. In an embodiment in which a single-base extension reaction is performed, the synthesis reaction is terminated with only a single-base extension by using, for example, a dideoxynucleotide (ddNTP) as the nucleotide (substrate) to be incorporated. In an embodiment in which a multiple-base extension reaction is performed, a combination of multiple substrates having fluorescent dyes is used as the nucleotides (substrates) to be incorporated. Substrates having fluorescent dyes are used as substrates for the base extension reaction, and the substrates include substrates having different fluorescent dyes for different target nucleic acids. Fluorescent dyes are useful for easily detecting whether or not a substrate has been incorporated, or for determining the type of incorporated base, and fluorescent dyes known in the art can be used (for example, but not limited to, fluorescein, fluorescein isothiocyanate (FITC), sulforhodamine (TR), tetramethylrhodamine (TRITC), carboxy-X-rhodamine (ROX), carboxytetramethylrhodamine (TAMRA), NED, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), rhodamine 110 (R110), rhodamine 6G (R6G), Alexa Fluor (registered trademark) system, Texas red, Cy system, etc.). There are no particular limitations on the type and introduction method of such fluorescent dyes, and various conventionally known means can be used.

[0045] After the base extension reaction, the reaction product obtained is subjected to electrophoresis for analysis. The electrophoresis is not particularly limited as long as it is a measurement method capable of performing fragment analysis by electrophoresis, and for example, electrophoresis in a microchannel such as capillary electrophoresis (CE) or MEMS (Micro-Electro-Mechanical Systems) can be used. Electrophoresis, for example, CE, is a method for separating introduced components based on differences in mobility based on charge, size, shape, and the like. Based on the mobility, the type of target nucleic acid (based on the type of primer) can be identified. In addition, based on the signal of the fluorescent dye, the presence or absence of the target nucleic acid or the type of a specific base in the target nucleic acid (based on the type of substrate incorporated by the base extension reaction), for example, wild type and mutant type, can be distinguished.

[0046] In the present invention, the long-chain ssDNA primer used in the gene analysis method has a longer base length than ssDNA primers synthesized by conventional chemical synthesis, and therefore can detect up to 700 bases, which is the detection limit of the electrophoresis device. This broadens the range that can be used to distinguish different target nucleic acids based on differences in base length, and as a result, it becomes possible to simultaneously detect a larger number of target nucleic acids (in one run) with high sensitivity.

[0047] In yet another aspect, the present invention provides a kit for use in a method for synthesizing a long single-stranded DNA (ssDNA) primer having a length of 100 to 700 bases, comprising: a forward primer to which a length determination sequence that determines the length of the ssDNA primer has been added; and A template containing a non-target sequence that is non-specific to the target nucleic acid Including, The method includes using the forward primer together with a reverse primer to which a sequence complementary to a target recognition sequence has been added at the 5' end to amplify a region containing a non-target sequence in the template to prepare a double-stranded DNA fragment containing the chain length determination sequence, the non-target sequence, and the target recognition sequence, and recovering from the double-stranded DNA fragment an ssDNA having the target recognition sequence at its 3' end.

[0048] The kit of the present invention provides a forward primer and a template used for synthesizing a long ssDNA primer as a kit, thereby enabling a user to synthesize a long ssDNA primer more quickly and simply by preparing a reverse primer according to the target nucleic acid of interest.

[0049] In one embodiment, the forward primer further has a nuclease-resistant structure at the 5'-end, and the method includes recovering ssDNA having a target recognition sequence at the 3'-end from the double-stranded DNA fragment using a nuclease having DNA degradation activity from the 5'-end to the 3'-end. The nuclease-resistant structure and the nuclease are as described above.

[0050] In one embodiment, the forward primer can include a plurality of primers to which chain length determining sequences of different lengths are added. By using the forward primers to which chain length determining sequences of different lengths are added in combination with reverse primers to which sequences complementary to different target recognition sequences are added, it is possible to synthesize a plurality of ssDNA primers of different lengths for different target recognition sequences.

[0051] The kit of the present invention may further include a reverse primer, in which case the user of the kit adds a sequence complementary to the target recognition sequence to the 5' end of the reverse primer for use in the amplification reaction.

[0052] The kit according to the present invention may contain, in addition to the above-mentioned components, a buffer constituting a reaction solution, enzymes (polymerase, nuclease, etc.), a substrate, and the like.

[0053] The present invention will be specifically described below by way of examples. However, these examples are provided merely for the purpose of explaining the present invention and are not intended to limit or restrict the scope of the invention disclosed in this application.

[0054] [Example 1] In this example, lambda exonuclease was used as an enzyme for recovering long-chain ssDNA primers in the method described in embodiment 3. Lambda exonuclease has DNA degradation activity from the 5' end to the 3' end, and is known to have the property of specifically degrading DNA strands whose 5' ends are phosphorylated. Therefore, in this example, the 5' end of the forward primer was made dephosphorylated and the 5' end of the reverse primer was made phosphorylated to resist nuclease degradation. Due to the difference in the structure of the 5' end, a long-chain ssDNA primer suitable for detection of the target base by the fluorescent-labeled base extension reaction method can be obtained by the method described in FIG. 3.

[0055] FIG. 4 shows the detailed arrangement of the phosphate-modified primer in the target nucleic acid used in the experiment of this embodiment. EGFR L858, a driver mutation in lung cancer, is a single-base substitution mutation in which the wild-type T base is changed to a G base. As shown in (401), the 5' end of the phosphate-modified primer (EGFR L858 Fw_RCP) (5'-GCCCAAAATCTGTGATCTTGACATGCTG-3': SEQ ID NO: 1) was arranged at the base position (indicated by an asterisk) immediately before the base substitution position as a primer for detecting this base mutation. Similarly, as shown in (402), the 5' end of the phosphate-modified primer (EGFR T790 Fw_RCP) (5'-TGATGAGCTGCACGGTGGAGGTGAGG-3': SEQ ID NO: 2) was arranged at the base position immediately before the single-base substitution mutation in which the wild-type C base is changed to a T base for EGFR L790, another driver mutation in lung cancer.

[0056] (403) is an example of the target base site of the amplified DNA product using the EGFR L858 Fw_RCP primer. The 5' end of the double-stranded DNA molecule is phosphate-modified (star), and by treating this DNA fragment with lambda exonuclease, which specifically degrades the phosphate-modified DNA strand, an ssDNA primer with the 3' end at the base immediately preceding the target is obtained.

[0057] In this experiment, PCR amplification was performed using plasmid DNA (pCR 2.1 topo) as template DNA, and the PCR amplification product in the synthesis of long-chain ssDNA primers was evaluated. Specifically, for the synthesis of ssDNA primers using the EGFR L858 Fw_RCP primer, plasmid DNA (pCR 2.1 topo) in which the EGFR L858 region was cloned was used as a template, and the EGFR L858 Fw_RCP primer (SEQ ID NO: 1) with its 5' end modified with phosphorylation was used as a reverse primer with a target recognition sequence, and the M13 F primer (5'-GTAAAACGACGGCCAG-3': SEQ ID NO: 3) that recognizes the base sequence of the plasmid vector was used as a forward primer to determine the chain length, and the 359 bp DNA chain (SEQ ID NO: 4) shown below was synthesized by PCR. In the following sequence, the part shown in a square frame is the recognition site of the forward primer, the part shown in italics and double underlined is the recognition site of the phosphoryl-modified primer, the underlined part is the base sequence of the cloned EGFR, and the other parts are the base sequences of the plasmid.

[0058] [ka]

[0059] For the synthesis of ssDNA primers using the EGFR T790 Fw_RCP primer, a plasmid DNA (pCR 2.1 topo) in which the EGFR T790 region was cloned was used as a template, and the EGFR T790 Fw_RCP primer (SEQ ID NO: 2) with its 5'-end modified with phosphorylation was used as a reverse primer having a target recognition sequence, and the M13 rev-150 primer (5'-GCTGGCACGACAGGTTTCCC-3': SEQ ID NO: 5) that recognizes the base sequence of the plasmid vector was used as a forward primer to determine the chain length. The 484 bp DNA chain (SEQ ID NO: 6) shown below was synthesized by PCR. In the following sequence, the part shown in a square frame is the recognition site of the forward primer, the part shown in italics and double underlined is the recognition site of the phosphoryl-modified primer, the underlined part is the base sequence of the cloned EGFR, and the other parts are the base sequences of the plasmid.

[0060] [ka]

[0061] Figure 5 shows the electrophoretic images of the amplified products obtained by PCR. The length of the amplified product of EGFR T790 was approximately 500 bp (Figure 5A), and the length of the amplified product of EGFR L858 was approximately 360 bp (Figure 5B). Based on the base sequence information of the template DNA used and the position information of the forward primer used in the amplification, the designed chain length of the long ssDNA primer targeting EGFR L858 was calculated to be 359 mer, and the designed chain length of the long ssDNA primer targeting EGFR T790 was calculated to be 484 mer, and it was confirmed that DNA fragments of the designed length were specifically obtained. These amplified DNAs were digested with lambda exonuclease and used in the subsequent experiments.

[0062] [Example 2] Using the long ssDNA primers (SEQ ID NOs: 4 and 6) synthesized in Example 1, the wild-type of each target gene was subjected to a fluorescently labeled single-base extension reaction using SNaPshot reagent (Thermo Fisher Scientific), and then analyzed using Seqstudio (Applied Biosystems).

[0063] The results are shown in Figure 6. In the test for EGFR T790, as shown in Figure 6B, a black waveform signal indicating the detection of the wild-type C base was observed at the 485-mer position. In the test for EGFR L858, as shown in Figure 6A, a red waveform signal indicating the detection of the wild-type T base was observed at the 360-mer position. The appearance position of each waveform was the base length of the designed primer sequence plus one base, and it was confirmed that these detection signals were the result of a one-base extension reaction.

[0064] [Example 3] In this example, a mixed sample of wild-type and mutant DNA fragments was prepared to verify the detection of mutations, and a base mutation detection test was performed by single-base extension reaction using the SNaPshot reagent. The mixed samples used contained 0%, 0.3%, 1%, 5%, or 10% mutant.

[0065] FIG. 7 shows the results of analyzing the SNaPshot reaction using the 359-mer EGFR L858 primer. A red waveform indicating the detection of the wild-type T base was observed at the 360-mer position (W=T). The mutant type of EGFR L858 added to the reaction system was a G base, and a blue waveform of the G base (M=G), in which differences in signal intensity were observed depending on the amount of the mutant mixed (0.3% to 10%), was observed before the wild-type waveform. The blue waveform that appears after the wild-type waveform is constant and does not reflect differences in the composition of the template, and is a noise signal independent of base detection. In addition, since the peak positions are different from the detection coordinates of the wild-type and mutant types, it does not interfere with base detection by the long-chain ssDNA of the present invention. Therefore, it was shown that the long-chain ssDNA primer synthesized in Example 1 can detect even a small amount of mutant type with high sensitivity.

[0066] In all the drawings for explaining the present embodiment, the same reference numerals are used for the components having the same functions, and repeated explanations are omitted as much as possible. Furthermore, the present invention is not to be interpreted as being limited to the description of the embodiment shown below. It is easily understood by those skilled in the art that the specific configuration can be changed without departing from the concept or purpose of the present invention.

[0067] 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.

[0068] As used herein, elements referred to in the singular are intended to include the plural unless the context clearly indicates otherwise.

[0069] All publications and patent documents cited in this specification are incorporated herein by reference in their entirety as part of the description of this specification. [Explanation of symbols]

[0070] 101, 201, 301 Forward primer that determines the length of the ssDNA primer 102, 202, 302 Reverse primer with target recognition sequence at the 3' end 103, 203, 303 DNA fragments containing non-target sequences 104, 204, 304 double-stranded DNA fragments 105, 205, 305 Long ssDNA primer 206 Biotin 307 Nuclease resistance at the 5' end [Sequence List Free Text]

[0071] SEQ ID NOs: 1-2: Synthetic oligonucleotides (1 base, phosphorylated) SEQ ID NOs: 3 and 5: synthetic oligonucleotides SEQ ID NOs: 4 and 6: synthetic polynucleotides

Claims

1. A method for synthesizing a long single-stranded DNA (ssDNA) primer having a length of 100 to 700 bases, comprising the steps of: a step of amplifying a region of a template containing a non-target sequence that is non-specific to the target nucleic acid, using a forward primer to which a length determination sequence that determines the length of the ssDNA primer has been added and a reverse primer to which a sequence complementary to a target recognition sequence has been added at the 5' end, thereby preparing a double-stranded DNA fragment containing the length determination sequence, the non-target sequence, and the target recognition sequence; a step of recovering ssDNA having the target recognition sequence at the 3' end from the double-stranded DNA fragment. The method includes:

2. the reverse primer further has a modification at the 5' end, The method according to claim 1, wherein in the recovery step, ssDNA having the target recognition sequence at its 3' end is recovered from the double-stranded DNA fragment by utilizing modification of the 5' end.

3. The method of claim 2 , wherein the 5′ end modification comprises a biotin modification.

4. the forward primer further has a nuclease resistant structure at its 5' end, The method according to claim 1, wherein in the recovery step, ssDNA having the target recognition sequence at the 3' end is recovered from the double-stranded DNA fragment using a nuclease having DNA degradation activity from the 5' end to the 3' end.

5. The method of claim 4, wherein the nuclease-resistant structure comprises a dephosphorylation structure and the nuclease comprises a lambda exonuclease.

6. 2. The method of claim 1, wherein the ssDNA primer is greater than 200 bases in length.

7. A reagent for use in genetic analysis by base extension reaction and electrophoresis, comprising a long ssDNA primer synthesized by the method of claim 4.

8. carrying out a base extension reaction on a test nucleic acid using a long ssDNA primer synthesized by the method of claim 1 and a substrate having a fluorescent dye; subjecting the reaction product of the base extension reaction to electrophoresis; detecting a target nucleic acid based on said electrophoretic mobility and said fluorescent dye. A genetic analysis method comprising:

9. The method of claim 8, wherein the long ssDNA primer comprises multiple long ssDNA primers for multiple target nucleic acids.

10. The method of claim 8 , wherein the target nucleic acid comprises a single nucleotide polymorphism (SNP).

11. A kit for use in a method for synthesizing a long single-stranded DNA (ssDNA) primer having a length of 100 to 700 bases, comprising: a forward primer to which a length determination sequence that determines the length of the ssDNA primer has been added; and A template containing a non-target sequence that is non-specific to the target nucleic acid Including, the method comprising: amplifying a region in the template containing a non-target sequence using the forward primer together with a reverse primer having a sequence complementary to a target recognition sequence added to the 5' end, thereby preparing a double-stranded DNA fragment containing the chain length determination sequence, the non-target sequence, and the target recognition sequence; and recovering from the double-stranded DNA fragment an ssDNA having the target recognition sequence at its 3' end.

12. the forward primer further has a nuclease resistant structure at its 5' end, The kit of claim 11, wherein the method comprises recovering ssDNA having the target recognition sequence at its 3' end from the double-stranded DNA fragment using a nuclease having DNA degradation activity from the 5' end to the 3' end.

13. The kit according to claim 11 , wherein the forward primer comprises a plurality of primers to which chain length determining sequences of different lengths have been added.

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