Long-chain ssdna primer for base detection

Abstract

The present invention pertains to a single-stranded DNA (ssDNA) primer having a chain length that exceeds 200 mer, a method for synthesizing the same, and a gene analysis method that uses the ssDNA primer. Specifically, the present invention pertains to a method for synthesizing a long-chain single-stranded DNA (ssDNA) primer that is 100-700 base long. The method comprises: a step for using a forward primer having a chain length determination sequence for determining the chain length of the ssDNA primer added thereto, and a reverse primer having a sequence complementary to the target recognition sequence added thereto at the 5' end, to amplify a region that includes a non-target sequence which is non-specific to the target nucleic acid in the template, thereby preparing a double-stranded DNA fragment that includes the chain length determination sequence, the non-target sequence, and the target recognition sequence; and a step for recovering ssDNA having the target recognition sequence at the 3' end from the double-stranded DNA fragment.

Description

Long ssDNA primer for base detection

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

[0002] There are various individual differences in genomes, and these differences in genome sequence are useful biomarkers for disease and drug response. Therefore, there is a need for low-cost detection of genome sequence variations in diagnostic and clinical settings.

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

[0004] 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 attaching mobility-altering labels to the primers, the signal detection position can be changed, allowing for simultaneous detection of multiple mutations. Depending on the target gene or mutation, it is also possible to detect the gene or mutation by extending multiple bases rather than by single-base extension. Non-Patent Document 1 reports simultaneous detection of 26 plex (26 types of mutations), but the analytical range used is a ~120 bp region of the analytical range of a capillary electrophoresis (CE) sequencer. While CE sequencers have the precision to resolve single-base differences in length in the range of 50–600 bp (up to 700 bp depending on the instrument), existing fluorescent-labeled single-base extension reaction methods only utilize a portion of the detection range of the CE sequencer.

[0005] One of the reasons for the limited detection range of CE sequencers is the difficulty of chemically synthesizing oligo DNAs used as primers that are longer than 200 bp. While mobility correction using labels other than nucleic acids, as in Meaghe et al. (Non-Patent Document 2), is effective in solving this problem, it is not easy to synthesize many types of these polymers with high purity and polymerization degrees.

[0006] Furthermore, in base measurement using the fluorescently labeled single-base extension reaction method, the PCR product used as a template can cause noise signals. TM This is described in the troubleshooting section of the protocol provided with the Multiplex Kit. The target amplification size for multiplex PCR is generally around 200 bp (Non-Patent Document 3). The strand length of cell-free DNA contained in liquid biopsies, which will be an important target sample for future genetic testing, is approximately 167 bp (Non-Patent Document 4), and PCR amplification products using liquid biopsies as samples will have fragment lengths of around 200 bp. In the fluorescent-labeled single-base extension reaction method, primers near the limit of chemical synthesis length overlap with the noise signal region derived from the PCR amplification product, making it difficult to use.

[0007] U.S. Patent No. 5,888,819

[0008] Coutinho1 et, al., PLOS ONE, 9(3):e923292, 2014Meagher et, al., Anal. Chem., 79(5):1848-1854, 2007Thornton B. et al., Biochem Mol Biol Educ. 39(2):145-154, 2011Lapin, M. et al., J. Transl. Med. 16(1):300, 2018

[0009] To increase the number of multiplexes (number of items that can be detected simultaneously) when measuring bases using the fluorescently 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 PCR product template, 200 bp.

[0010] As a result of intensive research into the synthesis of long ssDNA primers with the aim of diversifying existing fluorescently labeled single-base extension reaction methods (and even fluorescently labeled base extension reaction methods not limited to single-base extension reactions), we discovered a method for synthesizing long 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 relates to a method for synthesizing a long single-stranded DNA (ssDNA) primer of 100 to 700 bases in length, the method comprising: preparing double-stranded DNA fragments containing the length determination sequence, the non-target sequence, and the target recognition sequence by amplifying a region of a template containing a non-target sequence that is non-specific to a 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; and recovering ssDNA having the target recognition sequence at the 3' end from the double-stranded DNA fragments.

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

[0013] In another aspect, the present invention relates to a genetic analysis method comprising: a step of performing a base extension reaction on a test nucleic acid using a long-chain ssDNA primer synthesized by the above-described method and a substrate for a base extension reaction having a fluorescent dye; a step of subjecting a reaction product of the base extension reaction to electrophoresis; and a step of detecting a target nucleic acid based on the electrophoretic mobility and the fluorescent dye.

[0014] In yet another aspect, the present invention relates to a kit for use in a method for synthesizing a long single-stranded DNA (ssDNA) primer of 100 to 700 bases in length, comprising: a forward primer having a chain length determination sequence added thereto that determines the chain length of the ssDNA primer; and a template comprising a non-target sequence that is non-specific to a target nucleic acid, wherein the method comprises using the forward primer together with a reverse primer having a sequence complementary to the target recognition sequence added to its 5' end to amplify a region of the template that contains the non-target sequence, thereby preparing double-stranded DNA fragments that contain the chain length determination sequence, the non-target sequence, and the target recognition sequence, and recovering from the double-stranded DNA fragments ssDNA that has the target recognition sequence at its 3' end.

[0015] This specification includes the disclosure of Japanese Patent Application No. 2023-172527, filed October 4, 2023, which is the basis of priority for this application.

[0016] The use of long-chain base extension primers for detecting target nucleic acids has enabled the use of a wider detection chain length range for instruments used in fluorescent-labeled base extension reactions. This expansion of the detection chain length range increases the number of multiplex detections in one run, i.e., the number of types of target nucleic acids that can be detected simultaneously.

[0017] 1 shows an outline of a method for synthesizing a long-chain ssDNA primer. 2 shows an outline of a method for synthesizing a long-chain ssDNA primer using a modified reverse primer. 3 shows an outline of a method for synthesizing a long-chain ssDNA primer using a nuclease-resistant forward primer. 4 shows a schematic diagram showing the location of the phosphate-modified primer in the target nucleic acid used in the experiment of Example 1. 5 shows a photograph showing an electrophoretic image of an amplification product obtained by PCR using the primers designed in Example 1. 6 shows the results obtained by a fluorescently labeled single-base extension reaction (SNaPshot reaction) using a long-chain ssDNA primer in Example 2. 7 shows the results of analyzing a fluorescently labeled single-base extension reaction (SNaPshot reaction) using a long-chain ssDNA primer in Example 3.

[0018] The present invention relates to a method and kit for synthesizing long single-stranded DNA (ssDNA) primers, particularly long ssDNA primers for use in fluorescently labeled base extension reactions, as well as the resulting long ssDNA primers and their uses. The ssDNA primer has a sequence (target recognition sequence) at its 3' end that specifically binds to the sequence immediately preceding the target base in a target nucleic acid. As a result, after binding to the target nucleic acid, the primer can incorporate a base corresponding to the target base into the 3' end of the primer via a base extension reaction, thereby extending the primer. The base length of the ssDNA primer affects the base length (size) of the reactant in the base extension reaction, and therefore determines the electrophoretic mobility of the ssDNA primer. In fluorescently labeled base extension reactions, the target base can be detected based on the type of incorporated base and its electrophoretic mobility. Therefore, it is necessary to appropriately design the base sequence (target recognition sequence) and base length of the ssDNA primer.

[0019] Generally, there is no limit to the base length of the ssDNA primer as long as it has a target recognition sequence. In the present invention, a long-chain ssDNA primer having 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 electrophoresis equipment, for example, 700 bases or less, but varies depending on the equipment used.

[0020] In one aspect, the present invention provides a method for synthesizing a long single-stranded DNA (ssDNA) primer of 100 to 700 bases in length, the method comprising: preparing double-stranded DNA fragments containing the length determination sequence, the non-target sequence, and the target recognition sequence by amplifying a region of a template containing a non-target sequence that is non-specific to a 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; and recovering ssDNA having the target recognition sequence at the 3' end from the double-stranded DNA fragments.

[0021] First, a region of the template containing a non-target sequence is amplified using a forward primer and a reverse primer, each of which is designed to contain a sequence (PCR recognition sequence) that anneals to the corresponding region of the template.

[0022] Design techniques for forward and reverse primers are well known in the art. For example, forward and reverse primers usable in the present invention are designed to contain sequences (PCR recognition sequences) that satisfy conditions for specific annealing, such as 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. Known primer design software can be used to confirm the Tm. The designed primers can be chemically synthesized using known oligonucleotide synthesis techniques, but are typically synthesized using a commercially available chemical synthesizer.

[0023] 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. Thus, 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 the 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 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 contain a target-specific sequence. The specific sequence of the chain length determination sequence can be appropriately designed by one skilled in the art so that the chain length determination sequence does not form a hairpin structure.

[0024] In addition to a sequence (PCR recognition sequence) that anneals to the template, the reverse primer also contains a sequence complementary to the target recognition sequence at its 5' end. The target recognition sequence is a sequence specific to the target nucleic acid containing the base to be detected by the fluorescently labeled base extension reaction method, specifically, 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, approximately 20 bases. The double-stranded DNA fragment amplified by this reverse primer will contain a DNA strand having the target recognition sequence at its 3' end.

[0025] 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. Those skilled in the art can appropriately design the specific sequence of the non-target sequence so that the non-target sequence portion does not form a hairpin structure in the ssDNA primer.

[0026] 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.

[0027] Amplification produces double-stranded DNA fragments containing a length-determining sequence, a non-target sequence, and a target recognition sequence. From these double-stranded DNA fragments, ssDNA with the target recognition sequence at the 3' end is recovered. This allows the synthesis of ssDNA primers of the desired base length (100-700 bases).

[0028] [Embodiment 1] Figure 1 shows an overview of a method for synthesizing a long ssDNA primer for use in a fluorescently labeled base extension reaction. A set of primers amplifies a non-target sequence that is not complementary to the target nucleic acid. Specifically, a DNA fragment 103 containing a non-target sequence is amplified using a forward primer 101 (Length control_1) (0-100 mer) that determines the length of the ssDNA primer (including a mobility design portion, a length-determining sequence), and a reverse primer 102 (Target specific) (>10 mer) that has a sequence complementary to a target recognition sequence at its 5' end. A DNA strand containing the target recognition sequence at its 3' end is recovered from the double-stranded DNA fragment 104 obtained by amplification, yielding a long ssDNA primer 105 for detecting the target base by a fluorescently labeled base extension reaction.

[0029] [Embodiment 2] Figure 2 is a schematic diagram of a method for synthesizing a long ssDNA primer, in which a reverse primer 202 is pre-modified with biotin 206. 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) bearing a biotin modification 206 and a sequence complementary to a target recognition sequence at its 5' end. In the double-stranded DNA fragment 204 obtained by amplification, the DNA strand bearing the target recognition sequence at its 5' end retains the biotin modification 206. The double-stranded DNA fragment 204 is adsorbed onto streptavidin-modified beads and subjected to thermal denaturation. DNA strands 205 that are not adsorbed to the beads due to their lack of biotin modification are selectively recovered from the beads. This process yields a long ssDNA primer 205 for detecting target bases using a fluorescently labeled base extension reaction.

[0030] Therefore, in one embodiment, in the method of the present invention, the reverse primer further has a modification at its 5' end, and in the recovery step, the 5' end modification is used to recover ssDNA having a target recognition sequence at its 3' end from the double-stranded DNA fragment.

[0031] The modification of the 5' end of the reverse primer is not limited as long as it can be used to distinguish and recover ssDNA in the recovery step, and examples include biotin modification, digoxigenin modification, amino group modification, and thiol modification. This makes it possible to simply and efficiently recover ssDNA primers having a target recognition sequence at their 3' ends using the 5' end modification.

[0032] [Embodiment 3] Figure 3 is a schematic diagram of a method for synthesizing a long ssDNA primer using a forward primer 301 with a nuclease-resistant 307 at its 5' end. A DNA fragment 303 containing a non-target sequence is amplified using a forward primer 301 (Length control_1) with a nuclease-resistant 307 at its 5' end and a reverse primer 302 (Target specific) with a sequence complementary to a target recognition sequence at its 5' end. In the double-stranded DNA fragment 304 obtained by amplification, the 5' end of the DNA strand containing the target recognition sequence at its 3' end retains the nuclease-resistant 307. The double-stranded DNA fragment 304 is degraded with lambda exonuclease, which has DNA degradation activity from the 5' end to the 3' end, selectively leaving behind a DNA strand 305 with nuclease resistance at its 5' end. The DNA strand obtained by this process has a target recognition sequence at the 3' end, and the nuclease resistance at the 5' end ensures that the ssDNA strand length is maintained even after treatment with DNase. This makes it a long-chain ssDNA primer suitable for detecting target bases using the fluorescently labeled base extension reaction method.

[0033] Therefore, in one embodiment, in the method of the present invention, the forward primer further has a nuclease-resistant structure at the 5' end, and in the recovery step, a nuclease having DNA degradation activity from the 5' end to the 3' end is used to recover ssDNA having a target recognition sequence at the 3' end from the double-stranded DNA fragment.

[0034] Nuclease resistance means resistance to 5' to 3' degradation by nucleases. The nuclease-resistant structure of the forward primer is not particularly limited as long as it is a structure that is not degraded by nucleases that have 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 that has a target recognition sequence at the 3' end.

[0035] The method of the present invention allows the synthesis of long ssDNA primers having a desired base length. By adjusting the base length depending on the type of target recognition sequence, it is also possible to synthesize long ssDNA primers for each target recognition sequence, which enable simultaneous detection of multiple target recognition sequences by electrophoresis.

[0036] 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.

[0037] As described above, the long-chain ssDNA primer of the present invention comprises a length-determining sequence, a non-target sequence, and a target recognition sequence at the 3' end, and can be used in fluorescently labeled base extension reactions (genetic analysis by base extension 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, maximizing the detection range of 50 to 700 bases in instruments used in fluorescently labeled base extension reactions (e.g., capillary electrophoresis devices). Therefore, the long-chain ssDNA primer of the present invention enables the simultaneous detection of a larger number of target nucleic acids with high sensitivity in fluorescently labeled base extension reactions. Here, the fluorescently labeled base extension reaction is preferably a fluorescently labeled single-base extension reaction (genetic analysis by single-base extension and electrophoresis).

[0038] In another aspect, the present invention provides a method for genetic analysis using a long-chain ssDNA primer synthesized by the method of the present invention. Specifically, the method for genetic analysis of the present invention includes the steps of: performing a base extension reaction on a test nucleic acid using the long-chain 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; and detecting a target nucleic acid based on the electrophoretic mobility and the fluorescent dye.

[0039] 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 an ssDNA primer (which may not be 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).

[0040] 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, as described, for example, in Non-Patent Documents 1 and 2 and Patent Document 1.

[0041] 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 the target nucleic acid in the presence of a substrate (dideoxynucleotide triphosphate: ddNTP) bound to a fluorescent dye.

[0042] In the present invention, a target nucleic acid refers to a nucleic acid containing a base sequence for which detection (and quantification) is desired. For example, the target nucleic acid contains a base sequence containing a genetic mutation, and the target nucleic acid is detected (and quantified) by distinguishing between the wild type and the mutant type. Examples of such genetic mutations include, but are not limited to, single nucleotide polymorphisms (SNPs), insertion mutations, and deletion mutations. Furthermore, the mutant type of the target nucleic acid may contain one type of mutation or multiple types of mutations. For example, there are genetic mutations in which the wild type and two or three types of mutations exist, and such multiple types of mutations can also be distinguishably detected (and quantified).

[0043] 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.Nucleic acid can be prepared by methods known in the art.

[0044] In the genetic analysis method, a base extension reaction is carried out using the above-mentioned ssDNA primer in the presence of a substrate containing a fluorescent dye. Base extension reactions are well 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 a DNA or RNA template, respectively.

[0045] 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, for example, a dideoxynucleotide (ddNTP) is used as the incorporated nucleotide (substrate), and the synthesis reaction is completed with only a single-base extension. In an embodiment in which a multi-base extension reaction is performed, a combination of multiple substrates having fluorescent dyes is used as the incorporated nucleotides (substrates). 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 a substrate has been incorporated or for determining the type of incorporated base. 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®, Texas Red, Cy, etc.). The type and method of introduction of such fluorescent dyes are not particularly limited, and various conventionally known methods can be used.

[0046] After the base extension reaction, the resulting reaction product is subjected to electrophoresis for analysis. Electrophoresis can be performed by any measurement method capable of fragment analysis, including, for example, capillary electrophoresis (CE) and electrophoresis in microchannels such as MEMS (Micro-Electro-Mechanical Systems). Electrophoresis, such as CE, is a technique for separating introduced components based on differences in mobility, such as charge, size, and shape. Based on the mobility, the type of target nucleic acid (based on the type of primer) can be identified. Furthermore, based on the signal from the fluorescent dye, the presence or absence of the target nucleic acid or the type of specific base in the target nucleic acid (based on the type of substrate incorporated by the base extension reaction), such as wild-type or mutant, can be determined.

[0047] In the present invention, the long-chain ssDNA primers used in the genetic analysis method are longer than conventional chemically synthesized ssDNA primers, allowing detection of up to 700 bases, the detection limit of electrophoresis equipment. This broadens the range of nucleic acids that can be used to distinguish between different target nucleic acids based on differences in base length, and as a result, allows for the simultaneous detection of a larger number of target nucleic acids (in a single run) with high sensitivity.

[0048] In yet another aspect, the present invention provides a kit for use in a method for synthesizing a long single-stranded DNA (ssDNA) primer of 100 to 700 bases in length, comprising: a forward primer having a chain length determination sequence added thereto that determines the chain length of the ssDNA primer; and a template comprising a non-target sequence that is non-specific to a target nucleic acid, wherein the method comprises using the forward primer together with a reverse primer having a sequence complementary to the target recognition sequence added to its 5' end to amplify a region of the template that contains the non-target sequence, thereby preparing double-stranded DNA fragments that contain the chain length determination sequence, the non-target sequence, and the target recognition sequence, and recovering from the double-stranded DNA fragments ssDNA that has the target recognition sequence at its 3' end.

[0049] The kit of the present invention provides a forward primer and a template to be used for synthesizing a long-chain ssDNA primer as a kit, thereby enabling users to synthesize a long-chain ssDNA primer more quickly and easily by simply preparing a reverse primer according to the target nucleic acid of interest.

[0050] In one embodiment, the forward primer further has a nuclease-resistant structure at its 5'-end, and the method includes recovering ssDNA having a target recognition sequence at its 3'-end from the double-stranded DNA fragment using a nuclease having 5'- to 3'-end DNA degradation activity. The nuclease-resistant structure and the nuclease are as described above.

[0051] In one embodiment, the forward primer can include multiple primers to which chain length determining sequences of different lengths have been added. By using forward primers to which chain length determining sequences of different lengths have been added in combination with reverse primers to which sequences complementary to different target recognition sequences have been added, it is possible to synthesize multiple ssDNA primers of different lengths for different target recognition sequences.

[0052] The kit of the present invention may further comprise 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.

[0053] In addition to the above components, the kit according to the present invention may also contain a buffer, enzymes (polymerase, nuclease, etc.), substrates, etc. that constitute a reaction solution.

[0054] The present invention will be specifically described below by way of examples, but these examples are provided merely to illustrate the present invention and are not intended to limit or restrict the scope of the invention disclosed in this application.

[0055] Example 1 In this example, lambda exonuclease was used as the enzyme for recovering long ssDNA primers in the method described in Example 3. Lambda exonuclease has 5'- to 3'-terminal DNA degradation activity and is known to specifically degrade DNA strands phosphorylated at the 5' end. Therefore, in this example, the 5' end of the forward primer was dephosphorylated and the 5' end of the reverse primer was phosphorylated to resist nuclease degradation. Due to the difference in the 5' end structure, long ssDNA primers suitable for detection of target bases by fluorescently labeled base extension reaction can be obtained using the method described in Figure 3.

[0056] Figure 4 shows the detailed location of the phosphate-modified primers in the target nucleic acid used in the experiments of this example. EGFR L858, a driver mutation in lung cancer, is a single-base substitution mutation in which a wild-type T base is changed to a G base. As shown in (401), the 5' end of a phosphate-modified primer (EGFR L858 Fw_RCP) (5'-GCCCAAAATCTGTGATCTTGACATGCTG-3': SEQ ID NO: 1) was positioned at the base position immediately preceding the base substitution (indicated by an asterisk) to detect this base mutation. Similarly, as shown in (402), the 5' end of a phosphate-modified primer (EGFR T790 Fw_RCP) (5'-TGATGAGCTGCACGGTGGAGGTGAGG-3': SEQ ID NO: 2) was positioned at the base position immediately preceding the single-base substitution mutation in which a wild-type C base is changed to a T base for EGFR L790, another driver mutation in lung cancer.

[0057] (403) shows 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 (marked with an asterisk). 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.

[0058] In this experiment, PCR amplification was performed using plasmid DNA (pCR 2.1 topo) as the template DNA, and the PCR amplification products obtained by long-chain ssDNA primer synthesis were evaluated. Specifically, the EGFR L858 Fw_RCP primer was synthesized using plasmid DNA (pCR 2.1 topo) cloned with the EGFR L858 region as the template. The EGFR L858 Fw_RCP primer (SEQ ID NO: 1) with a phosphate-modified 5' end served as the reverse primer with the target recognition sequence, and the M13 F primer (5'-GTAAAACGACGGCCAG-3'; SEQ ID NO: 3) recognized the nucleotide sequence of the plasmid vector as the forward primer to determine the strand length. The 359 bp DNA strand shown below (SEQ ID NO: 4) was synthesized by PCR. In the following sequence, the boxed portion represents the recognition site for the forward primer, the italicized and double-underlined portion represents the recognition site for the phosphate-modified primer, the underlined portion represents the nucleotide sequence of the cloned EGFR, and the remaining portion represents the nucleotide sequence of the plasmid.

[0059]

[0060] To synthesize the ssDNA primer using the EGFR T790 Fw_RCP primer, plasmid DNA (pCR 2.1 topo) into which the EGFR T790 region had been cloned was used as a template. The EGFR T790 Fw_RCP primer (SEQ ID NO: 2) with its 5'-phosphate-modified end was used as a reverse primer with a target recognition sequence, and the M13 rev-150 primer (5'-GCTGGCACGACAGGTTTCCC-3': SEQ ID NO: 5) that recognizes the nucleotide sequence of the plasmid vector was used as a forward primer to determine the chain length. The 484 bp DNA strand shown below (SEQ ID NO: 6) was synthesized by PCR. In the following sequence, the boxed portion is the recognition site of the forward primer, the italicized and double-underlined portion is the recognition site of the phosphate-modified primer, the underlined portion is the nucleotide sequence of the cloned EGFR, and the remaining portion is the nucleotide sequence of the plasmid.

[0061]

[0062] Figure 5 shows electrophoretic images of the PCR amplification products. The length of the EGFR T790 amplification product was approximately 500 bp (Figure 5A), and the length of the EGFR L858 amplification product 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 lengths of the long ssDNA primers targeting EGFR L858 and EGFR T790 were calculated to be 359 mer and 484 mer, respectively, confirming that DNA fragments of the designed lengths were specifically obtained. These amplified DNAs were digested with lambda exonuclease and used in subsequent experiments.

[0063] [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).

[0064] The results are shown in Figure 6. In the test on EGFR T790, a black waveform signal indicating the detection of the wild-type C base was observed at the 485-mer position, as shown in Figure 6B. In the test on EGFR L858, a red waveform signal indicating the detection of the wild-type T base was observed at the 360-mer position, as shown in Figure 6A. The appearance position of each waveform was one base longer than the length of the designed primer sequence, confirming that these detection signals were the result of a single-base extension reaction.

[0065] In this example, to verify mutation detection, mixed samples of wild-type and mutant DNA fragments were prepared and subjected to a base mutation detection test by single-base extension reaction using the SNaPshot reagent. The mixed samples used contained 0%, 0.3%, 1%, 5%, or 10% mutant DNA.

[0066] Figure 7 shows the results of an SNaPshot reaction using a 359-mer EGFR L858 primer. A red waveform indicating the detection of a wild-type T base was observed at the 360-mer position (W = T). The mutant form of EGFR L858 added to the reaction system was a G base. A blue waveform (M = G) corresponding to the G base, whose signal intensity varied depending on the amount of mutant (0.3% to 10%), was observed before the wild-type waveform. The blue waveform appearing after the wild-type waveform is constant and does not reflect differences in template composition; it is a noise signal independent of base detection. Furthermore, since the peak positions are different from the detection coordinates of the wild-type and mutant forms, it does not interfere with base detection using the long-chain ssDNA of the present invention. Therefore, it was demonstrated that the long-chain ssDNA primer synthesized in Example 1 can detect even small amounts of mutant forms with high sensitivity.

[0067] In all the drawings for explaining the present embodiment, the same reference numerals are used to designate components having the same functions, and repeated explanations are omitted as much as possible. Furthermore, the present invention should not be interpreted as being limited to the description of the following embodiment. Those skilled in the art will easily understand that the specific configuration can be changed within the scope of the idea or purpose of the present invention.

[0068] 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.

[0069] As used herein, elements referred to in the singular are intended to include the plural unless the context clearly indicates otherwise.

[0070] The publications and patent documents cited in this specification are incorporated herein by reference in their entirety.

[0071] 101, 201, 301 Forward primer that determines the length of the ssDNA primer 102, 202, 302 Reverse primer with a target recognition sequence at the 3' end 103, 203, 303 DNA fragment containing a non-target sequence 104, 204, 304 Double-stranded DNA fragment 105, 205, 305 Long ssDNA primer 206 Biotin 307 Nuclease resistance at the 5' end

[0072] 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: preparing a double-stranded DNA fragment containing the length determination sequence, the non-target sequence, and the target recognition sequence, by amplifying a region in a template containing a non-target sequence that is non-specific to a 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; and recovering from the double-stranded DNA fragment an ssDNA having the target recognition sequence at the 3' end.

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

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

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

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

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

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

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

9. The method of claim 8, wherein the long ssDNA primers comprise 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, the kit comprising: a forward primer having a length determination sequence added thereto that determines the length of the ssDNA primer; and a template comprising a non-target sequence that is non-specific to a target nucleic acid, the method comprising: using the forward primer together with a reverse primer having a sequence complementary to a target recognition sequence added to the 5' end to amplify a region in the template that contains the non-target sequence, thereby preparing a double-stranded DNA fragment that contains the 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 kit of claim 11, wherein the forward primer further has a nuclease-resistant structure at the 5' end, and the method comprises recovering ssDNA having the 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.

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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