Primers for selective amplification of trace amounts of target sequences

Abstract

This invention discloses multipart amplification primers that distinguish between mutant DNA target sequences and closely related wild-type DNA target sequences. Furthermore, it discloses a method for detecting low-copy-number targets in a qPCR reaction using the disclosed primers.

Description

Technical Field

[0001] The present invention relates to nucleic acid amplification primers useful for selectively amplifying and / or detecting mutant DNA target sequences with extremely low copy numbers contained in samples such as liquid biopsies, which have an excessive amount of wild-type target DNA sequences, for diagnostic purposes. The primers are particularly useful for the specific and selective amplification and / or detection of trace target sequences that differ from a very abundant sequence by only one nucleotide. A method for amplifying and / or detecting trace nucleic acid targets in a qPCR reaction using the primers, as well as primer design and kits, are further disclosed.

Background Art

[0002] In order to reliably detect and quantify a target in a sample containing a low concentration of a target nucleic acid sequence, selective amplification of the target using polymerase chain reaction (PCR) and real-time detection (RT) of the generated amplification products (amplicons) may be required by a method known as RT-qPCR. The selection of specific primers containing short DNA sequences complementary to the target sequence is important to ensure specific amplification of the target while preventing non-specific amplification of related sequences contained in the sample. Therefore, the primer sequence is designed to be complementary to the target, resulting in hybridization of the primer to the target DNA sequence and its amplification product. By selecting a specific annealing temperature, annealing to (almost) completely complementary target sequences may be prioritized, and annealing of specific primers to related target sequences may be prevented. However, when using conventional DNA primers, specific amplification of very similar sequences that differ by only one or two nucleotides can be difficult.

[0003] Distinguishing between mutant and wild-type sequences based solely on single nucleotide polymorphisms (SNPs) is particularly challenging. While designing amplification primers that are perfectly complementary to the mutant DNA target sequence may favor the amplification of the corresponding target, wild-type sequences may also be amplified due to their very similar properties, such as annealing temperature and broad base complementarity. Unwanted amplification of wild-type DNA target sequences may be less efficient compared to the intended mutant DNA target sequence. However, detection and quantification of mutant and wild-type target sequences become unreliable.

[0004] This poses a particular diagnostic problem when detecting mutant DNA target sequences in samples overwhelmingly containing wild-type DNA target sequences is crucial. Due to the insufficient amplification selectivity provided by conventional primer designs, the presence of wild-type DNA sequences masks mutant DNA target sequences with low copy numbers.

[0005] One example of these challenges stemming from the low selectivity of conventional primer design is the identification of mutant DNA target sequences in circulating cell-free DNA. Circulating cell-free DNA (cfDNA) has been shown to have reasonable prognostic and diagnostic potential in several pathological conditions, including cancer, sepsis, and autoimmune diseases (e.g., systemic lupus erythematosus (SLE)). Cancer is one of the deadliest diseases and is projected to account for a significant proportion of deaths in an aging society. Early detection of cancer is associated with improved health and increased survival rates. While cancer symptoms can develop in the later stages of the disease, numerous approaches are available to detect cancer before symptoms appear. For example, broad screening is common in certain age groups, allowing for early identification of cancerous tissue and initiation of treatment. However, current diagnostic methods are insufficient in terms of cost-effectively and rapidly identifying early-stage cancer.

[0006] Cancer patients typically have high concentrations of cfDNA in their serum or plasma as a result of cellular necrosis or apoptosis, because tumor cells divide faster than normal cells and release cfDNA at a high rate (Sorenson et al., 1994; Vasioukhin et al., 1994; Raja et al., 2018). Fractions of cfDNA derived from tumor cells are called circulating tumor DNA (ctDNA) (Leon et al., 1977; Shu et al., 2017). In recent years, both cfDNA and ctDNA have attracted increasing attention as novel blood biomarkers. This is because their predictive and prognostic value has been suggested by the quantification and dynamic analysis of cfDNA (Diehl et al., 2008) and molecular profiling of ctDNA (Iizuka et al., 2006; Tokuhisa et al., 2007). Several liquid biopsy tests designed to identify cancer-specific mutations are recommended as companion diagnostics (CDx) tests by the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) to guide treatment decisions.

[0007] However, identifying mutant DNA target sequences, such as ctDNA, requires specific detection of mutant sequences and selective differentiation between mutants and wild-type targets. In recent years, isolating cfDNA and then sequencing it using next-generation sequencing (NGS) approaches has become a powerful tool not only for detecting target sequences but also for differentiating between mutants and wild-type sequences. However, because mutants have a lower copy number compared to wild-type sequences, extensive sample preparation and deep sequencing of samples are required, necessitating a considerable commitment of cost and time. Therefore, NGS approaches are currently insufficient to provide rapid, frequent, and cost-effective diagnostic screening.

[0008] Therefore, there is a great need for novel methods that can rapidly screen samples of mutant DNA target sequences while keeping costs low. qPCR detection is an established method for identifying and quantifying nucleic acid targets in clinical samples. However, conventional primers are insufficient in terms of amplification efficiency and selectivity when reliably identifying very low-concentration mutant DNA sequences that differ from wild-type alleles by only one or two bases. Innovations in primer design approaches have significantly improved amplification efficiency. For example, incorporating ribonucleic acid at selected positions within conventional primer designs reduces primer dimer formation, and therefore greatly improves PCR efficiency (e.g., International Publication 2009 / 004630).

[0009] Furthermore, the development of highly selective amplification primers addresses several needs for the specific and selective amplification of mutant DNA target sequences rather than closely related wild-type sequences. Numerous approaches to improving primer selectivity have been disclosed, including hairpin structure primers (e.g., International Publication 2000 / 71562), amplified refractory mutation system (ARMS) primers (Curr Protoc Hum Genet. 2001 May; Chapter 9: Unit 9.8. doi: 10.1002 / 0471142905.hg0908s07.), and multipart primers (e.g., superselective primers containing an internal sequence that is not complementary to the target sequence between two complementary target sequences (International Publication 2014 / 124290, International Publication 2017 / 176852, U.S. Patent Application Publication 2019 / 225999, International Publication 2021 / 067527)).

[0010] Furthermore, selective primers have been developed using various strategies, such as the stem-loop design disclosed in International Publication No. 2013 / 053852. European Patent No. 1185546 discloses a primer design including a stem and a loop, in which both the 3' arm sequence and the loop sequence are perfectly complementary to a selected priming region of the target DNA strand, and no single-stranded overhanging 3' end of the stem can nucleate with the complementary sequence. International Publication No. 2018 / 093898 relates to an SNP-specific hairpin primer characterized by a docking site and a hairpin. International Publication No. 2019 / 140298 relates to a primer design in which a target-specific section includes a stem-forming section that is hybridizable with the 5' terminal portion of the target-specific section and capable of forming a stem structure.

[0011] Despite significant progress, selectivity in PCR amplification remains a challenge. Therefore, there is a strong need for improved primers and / or alternative primers, as well as amplification methods, that enable selective amplification of mutant DNA target sequences, while closely related wild-type DNA target sequences are either not amplified or are amplified at very low concentrations. These would allow for cost-effective, rapid, and non-invasive diagnostics.

[0012] Therefore, the objective objective of the present invention was to develop novel primers and primer designs that enable specific and selective amplification of mutant DNA target sequences rather than closely related wild-type sequences, and are useful for quantitative assays, thereby improving the detection sensitivity and reliability of such approaches and enabling a cost-effective, comprehensive screening method. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] International Publication No. 2009 / 004630 [Patent Document 2] International Publication No. 2000 / 71562 [Patent Document 3] International Publication No. 2014 / 124290 [Patent Document 4] International Publication No. 2017 / 176852 [Patent Document 5] U.S. Patent Application Publication No. 2019 / 225999 [Patent Document 6] International Publication No. 2021 / 067527 [Patent Document 7] International Publication No. 2013 / 053852 [Patent Document 8] European Patent No. 1185546 [Patent Document 9] International Publication No. 2018 / 093898 [Patent Document 10] International Publication No. 2019 / 140298 [Non-patent literature]

[0014] [Non-Patent Document 1] Curr Protoc Hum Genet. 2001 May;Chapter 9:Unit 9.8. doi: 10.1002 / 0471142905.hg0908s07. [Overview of the Initiative]

[0015] A first aspect of the present invention is a multipart amplification primer that distinguishes between a mutant DNA target sequence and a closely related wild-type DNA target sequence, wherein the 5' to 3' direction is as follows: (i) A docking sequence that can form a hybrid of 15-40 nucleotides in length with a mutant DNA target sequence and a wild-type DNA target sequence, (ii) Hairpin sequences that cannot form hybrids with mutant DNA target sequences and wild-type DNA target sequences, (iii) Spacer arrangement, (iv) a palm sequence that is complementary to the mutant DNA target sequence and the wild-type DNA target sequence and can form a hybrid with a hairpin sequence having a length of 4 to 10 nucleotides, and (v) a mutation recognition sequence comprising a mutation-specific nucleotide located at the first or second position at the 5'-end of the sequence consisting of, or comprising, five consecutive DNA sequences of wherein the consecutive hairpin sequences, spacer sequences, and palm sequences form a stem-loop structure, the mutant target DNA sequence is completely complementary to the continuous sequence formed by the palm sequence and the mutation recognition sequence, and the wild-type DNA target sequence has a mismatch with respect to the continuous sequence having the mutation-specific nucleotide of the mutation recognition sequence, relating to a multi-part amplification primer.

[0016] In some embodiments, the docking sequence further comprises a dimer prevention nucleotide at the last position at the 3'-end, and the nucleotide does not pair with the mutation-specific nucleotide and / or any other nucleotide located at the 3'-end of the multi-part amplification primer. In some embodiments, the dimer prevention nucleotide is identical to the nucleotide located at the first position at the 5'-end of the mutation recognition sequence. In some embodiments, the dimer prevention nucleotide is a locked nucleic acid (LNA). In some embodiments, the mutation recognition sequence is not part of a stem-loop structure.

[0017] In some embodiments, the multi-part amplification primer further comprises at least one ribonucleotide. In a preferred embodiment, the at least one ribonucleotide is located within 10 nucleotides from the 3'-end of the primer, two ribonucleotides are not adjacent to each other, and the base at the 3'-end is a deoxyribonucleotide.

[0018] In some embodiments, the multipart amplification primer includes, or consists of, a nucleotide sequence selected from the group consisting of polynucleotides described in SEQ ID NOs. 9-22, 32-35, 38, 39, 44, and 46-50.

[0019] A further aspect of the present invention relates to a method for utilizing the multipart amplification primers of the present invention. Accordingly, a primer-dependent amplification and detection method is provided that, for each mutant DNA target sequence, at least one mutant DNA target sequence can be amplified and detected in a sample in a mixture containing closely related wild-type DNA target sequences that differ from the mutant DNA target sequence by only one or two base pairs, the following steps: (a) Provide a sample containing at least one mutant and / or wild-type DNA target sequence, and for each mutant DNA target sequence, provide a pair of forward primers and reverse primers. (b) Prepare a primer-dependent amplification reaction mixture comprising, or consisting of, DNA polymerase, deoxyribonucleoside triphosphate, amplification buffer, other reagents necessary for amplification, the sample, and forward and reverse primer pairs for each mutant DNA target sequence of step (a). (c) The mixture obtained thereby is subjected to repeated cycles of primer-dependent amplification reactions under primer annealing conditions including primer annealing temperature, to amplify each mutant DNA target sequence contained in the sample, and (d) The amount of the amplification product obtained thereby is measured to detect at least one mutant DNA target sequence. including, consisting of, or substantially consisting of, The multipart amplification primer according to any one of the claims, wherein the forward primer and / or reverse primer of the primer pair for each mutant DNA target sequence are specific to the mutant DNA target sequence but have a mismatch with respect to the wild-type DNA target sequence, The docking sequence hybridizes with the mutant DNA target sequence and closely related wild-type DNA target sequence at the primer annealing temperature. Primer-dependent amplification and detection methods are further disclosed.

[0020] In some embodiments, the method according to the present invention can amplify and detect, in a sample, at least one mutant DNA target sequence, of which there are only 10 copies, in a mixture containing 10,000 copies of closely related wild-type DNA target sequences that differ from the mutant DNA target sequence by only one or two base pairs.

[0021] In some embodiments of this method, either the forward or reverse primer of the primer pair for each mutant DNA target sequence is a conventional primer. In some embodiments of this method, the docking sequence and palm sequence of the primer hybridize with either the mutant DNA target sequence or the wild-type DNA target sequence. In some embodiments, the probability that the multipart amplification primer / wild-type DNA target sequence hybrid is extended during the cycling is at least 100 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, and at least 700 times higher than the probability that the multipart amplification primer / mutant DNA target sequence hybrid is extended during the cycling. 0 times, at least 8,000 times, at least 9,000 times, at least 10,000 times, at least 20,000 times, at least 30,000 times, at least 40,000 times, at least 50,000 times, at least 60,000 times, at least 70,000 times, at least 80,000 times, at least 90,000 times, at least 100,000 times, at least 150,000 times, or at least 200,000 times lower, which is supported by at least, for example, a difference of 10 thermal cycles (ΔCq) (which corresponds to 1,000 times).

[0022] In some embodiments of this method, the forward primer and reverse primer of the forward primer and reverse primer pair are multipart amplification primers according to the present invention, where the forward primer has a mutation-specific nucleotide complementary to the mutation site contained in one of the two strands of the mutant DNA target sequence, and the reverse primer has a mutation-specific nucleotide complementary to the other mutant DNA target sequence of its base pair contained in the other of the two strands of the mutant DNA target sequence, so that one primer binds to the target strand and the other primer binds to the complementary target strand.

[0023] In some embodiments of this method, the closely related wild-type DNA target sequence differs from the mutant DNA target sequence by one base. In some embodiments, the docking sequence hybridizes with the mutant and wild-type DNA target sequences at the primer annealing temperature.

[0024] In some embodiments of this method, the primer-dependent amplification mixture of step (b) further comprises a homogeneous fluorescence detection means for detecting the amplification product, and step (d) includes detecting the at least one mutant DNA target sequence by measuring the fluorescence intensity emitted from the homogeneous fluorescence detection means. In some embodiments, the primer-dependent amplification and detection method is polymerase chain reaction (PCR), and the detection is real-time (RT) detection.

[0025] In some embodiments of this method, the sample includes cfDNA prepared from the serum and / or plasma of the subject.

[0026] Further embodiments relate to reagent kits for carrying out the methods of the present invention. In some embodiments, the kit comprises, or consists of, or substantially comprises, at least one pair of forward and reverse primers for one or more mutant DNA target sequences, dNTPs, primer-dependent polymerases, detection probes for each mutant DNA target, and other reagents necessary for amplification, in particular amplification buffers.

[0027] (definition) For convenience, the specific terms used in this specification, the examples, and the appended claims are summarized here.

[0028] In this specification, the singular forms "a," "an," and "the" include the plural form unless explicitly stated otherwise. For example, the term "sample" includes multiple samples.

[0029] Conventional primers are single-stranded oligonucleotides 15–40 nucleotides long (typically 20–30 nucleotides long) that are perfectly complementary to the intended target. Generally, any computer program can be used to design conventional PCR primers.

[0030] As used herein, the term “amplification” and its variations include any process for generating a number of copies or complements of at least a portion of a polynucleotide, which is generally referred to as the “template.” The template polynucleotide may be single-stranded or double-stranded. Amplification of a given template results in the generation of a group of polynucleotide amplification products, which are collectively referred to as “amplicons.” The polynucleotides of the amplicons may be single-stranded, double-stranded, or a mixture of both. Typically, the template contains a target sequence, and the resulting amplicons will contain polynucleotides having sequences that are either substantially identical to or substantially complementary to the target sequence. In some embodiments, the polynucleotides of a particular amplicon are substantially identical or substantially complementary to each other; or, in some embodiments, the polynucleotides contained in a given amplicon may have different nucleotide sequences. Amplification may be linear or exponential, and may involve the iterative and sequential replication of a given template, producing two or more amplification products. Some conventional amplification reactions involve a continuous and iterative cycle of nucleic acid synthesis based on a template, resulting in the formation of multiple daughter nucleotides. The daughter nucleotides contain at least a portion of the nucleotide sequence of the template and share at least some degree of identity (or complementarity) with the template's nucleotide sequence. In some embodiments, each step of nucleic acid synthesis, which may be referred to as an amplification "cycle," involves the creation of a free 3' end (e.g., by cleaving a single strand of dsDNA), thereby generating a primer, followed by a primer extension step; additional denaturation steps may also be included as appropriate, causing the template to partially or completely denaturate. In some embodiments, one round of amplification comprises a given number of repetitions of one amplification cycle. For example, an amplification round may comprise 5, 10, 15, 20, 25, 30, 35, 40, 50, or more repetitions of a particular cycle.In one exemplary embodiment, amplification includes any reaction in which a specific polynucleotide template is used in two consecutive cycles of nucleic acid synthesis. Synthesis may include template-dependent nucleic acid synthesis.

[0031] The term "primer" refers to a nucleic acid chain or oligonucleotide that can hybridize to a template nucleic acid and acts as a starting point for the incorporation of elongating nucleotides according to the sequence of the template nucleic acid during nucleic acid synthesis. "Elongating nucleotide" refers to any nucleotide that can be incorporated into the elongation product during amplification, i.e., DNA, RNA, or derivatives of DNA or RNA (which may include labels).

[0032] Hybridization, hybridizing, or annealing refers to the ability of fully or partially complementary nucleic acid strands to join together in a parallel or preferably antiparallel direction under specific hybridization conditions (e.g., annealing conditions) to form a stable double-stranded structure or region (sometimes called a "hybrid"), where the two strands constituting this structure or region are joined by hydrogen bonds. Hydrogen bonds are typically formed between adenine and tyrosine or uracil (A and T or U), or between cytosine and guanine (C and G), although other base pairs may be formed (see, for example, Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0033] As used herein, when two nucleic acid sequences form base pairs with each other at each position, they are “complementary” or “complementary” to each other. The term “complementary” is defined as a sequence that pairs with a given sequence based on standard base-pairing rules. For example, the single-stranded nucleotide sequence AGT is “complementary” to the other single-stranded sequence TCA. The terms “complementary” and the phrase “reverse complementary” are used herein interchangeably with respect to nucleic acids and are intended to define antisense nucleic acids.

[0034] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein. They refer to polymeric forms of nucleotides of any length: polynucleotides have any known or unknown three-dimensional structure and may perform any function. Examples of polynucleotides include, but are not limited to, coding or non-coding regions of genes or gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure may occur before or after polymer association. Nucleic acid sequences may be interrupted by non-nucleotide elements. Polynucleotides may be further modified, for example, by binding with labeling elements.

[0035] Unless otherwise specified, the term “nucleic acid” refers to any nucleic acid, whether single-stranded or double-stranded, such as ribonucleic acid, deoxyribonucleic acid, or xenonucleic acid.

[0036] The term "mutation site" refers to the sequence of target sequences in mutant and wild-type DNA that distinguishes the mutant from the wild-type. A mutation site may consist of one mismatched nucleotide (e.g., SNP) or multiple mismatched nucleotides.

[0037] As used herein, the term "identity," when applied to nucleic acids or polypeptides, refers to the degree of similarity between two or more nucleotide or polypeptide sequences. The ratio of "sequence identity" between two sequences can be determined by comparing two optimally aligned sequences in a comparison window, where the sequence portion within the comparison window may include additions or deletions (gaps) compared to a reference sequence (which does not include additions or deletions) in order to optimally align the two sequences. This ratio is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences, calculating the number of matching positions, dividing this number by the total number of positions in the comparison window, and then multiplying the result by 100 to obtain the ratio of sequence identity. A sequence that is identical in all positions compared to a reference sequence is considered identical to the reference sequence, and vice versa. Alignment of two or more sequences may be performed using any suitable computer program. For example, a widely used and accepted computer program for sequence alignment is CLUSTALW vl .6 (Thompson, et al. (1994) Nucl. Acids Res., 22: 4673-4680).

[0038] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In particular, unless otherwise specified, the terms used herein follow the definitions set forth in the Oxford Dictionary of Biochemistry and Molecular Biology (first edition 1997, revised edition 2000, reprinted edition 2003, ISBN 0 19 850673 2), published by Oxford University Press. [Modes for carrying out the invention]

[0039] The present invention relates to a multipart amplification primer for a primer-dependent amplification reaction capable of distinguishing between a mutant DNA target sequence and a closely related wild-type DNA target sequence, and is referred to as a switch primer. When hybridized to a mutant DNA target sequence and a closely related sequence, the switch primer significantly increases the selective amplification of the target mutant DNA target sequence and significantly inhibits the amplification of the wild-type DNA target sequence compared to conventional primer designs or other approaches known to those skilled in the art.

[0040] After amplification of the target mutant DNA target sequence contained in a sample such as a liquid biopsy, the amplicons are efficiently amplified exponentially, enabling real-time detection of the amplicon number and identification of the presence of the mutant DNA target sequence in the sample. Accordingly, the present invention discloses the design and properties of switch primers, as well as methods of use.

[0041] As disclosed in this specification and examples below, the switch primers are highly selective and can amplify the desired mutant DNA target sequence while suppressing the amplification of the wild-type DNA target sequence, even when the mutant and wild-type DNA target sequences are distinguished by single nucleotide polymorphisms (SNPs). Remarkably, the switch primers can identify the mutant DNA target sequence in a sample containing only 10 copies of the mutant DNA target sequence for every 10,000 copies of the wild-type DNA target sequence.

[0042] The multipart amplification primer (switch primer) according to the present invention has the following characteristics in the direction from 5' to 3': (i) A docking sequence that can form a hybrid of 15-40 nucleotides in length with a mutant DNA target sequence and a wild-type DNA target sequence, (ii) Hairpin arrangement, (iii) Spacer arrangement, (iv) Palm sequences complementary to the mutant DNA target sequence and the wild-type DNA target sequence, (v) A mutation recognition sequence containing the first or second mutation-specific nucleotide located at the 5' end of the sequence. It is characterized by consisting of, or containing, five consecutive DNA sequences (i) to (v).

[0043] A schematic diagram of an embodiment of the primer according to the present invention can be shown in Figure 1.

[0044] (Target sequence)

[0045] There is a strong industrial need to detect the presence and / or quantity of closely related sequences (e.g., DNA sequences) contained in a sample. For example, identifying mutant target DNA sequences and wild-type DNA target sequences (e.g., allele-specific mutations and wild-type alleles that are precursors to cancer) in a sample (e.g., liquid biopsy) enables disease detection and subsequent targeted therapy. Therefore, the mutant DNA target sequences described in this invention refer to sequences containing polynucleotides contained in a sample that require detection and / or quantification of their presence.

[0046] In some embodiments, the mutant and wild-type DNA target sequences are double-stranded DNA (dsDNA). In some embodiments, the mutant and wild-type DNA target sequences are single-stranded DNA (ssDNA).

[0047] (Mutation site)

[0048] The mutant DNA target sequence described in the present invention may differ from the wild-type target sequence, which may also be present in the sample, in a distinct mutation site. The mutation site refers to mismatched bases in the mutant and wild-type target DNA sequences, and their positions within the sequence. In some embodiments, the mutation site is contained within the DNA target sequence and consists of 1, 2, 3, or 4 mismatched nucleotides. In preferred embodiments, the mutation site consists of 2 mismatched bases, and more preferably 1 mismatched base (single nucleotide polymorphism (SNP)).

[0049] (Docking array (i))

[0050] The docking sequence (i) contained in the switch primer described in the present invention can hybridize with a common sequence (docking target site) of the mutant and wild-type DNA target sequences, allowing the primer to be positioned spatially close to the mutant site of the mutant / wild-type DNA target sequences, and enabling the identification of mutations with wild-type sequences, such as a single mismatched base (SNP). Hybridization may occur due to the overall or partial base complementarity of the docking sequence with respect to the docking target site contained in the mutant and wild-type DNA target sequences.

[0051] Adjusting the position of the switch primer described in this invention using a docking sequence to distinguish between mutant and wild-type DNA target sequences is essential for the primer's ability to favorably amplify the mutant DNA target while reducing the opportunity for amplification of the corresponding wild-type sequence. By positioning the docking sequence near the mutation site, a stable interaction between the primer's docking sequence and its target is possible before the remaining complementary portions of the multipart amplification primer described in this invention further bind. In some embodiments, the docking sequence is partially complementary to a common sequence (docking target site) of the mutant and wild-type DNA target sequences. In preferred embodiments, the docking sequence is fully complementary to the docking target site.

[0052] In some embodiments, the docking sequence is about 200 to about 5 nucleotides long, preferably about 100 to about 10 nucleotides long, more preferably about 80 to about 10 nucleotides long, even more preferably about 70 to about 15 nucleotides long, and most preferably about 50 to about 15 nucleotides long.

[0053] In some embodiments, the docking sequence can form a hybrid with the mutant DNA target sequence and the wild-type DNA target sequence that is about 10 to about 200 nucleotides long, more preferably about 10 to about 100 nucleotides long, even more preferably about 15 to about 50 nucleotides long, and most preferably about 15 to about 40 nucleotides long.

[0054] In some embodiments, the docking sequence hybridizes with a common docking target site located approximately 200 to 50 nucleotides downstream, preferably about 100 to 10 nucleotides, and most preferably about 50 to 10 nucleotides downstream, from the mutation site on the target strand of the mutant and wild-type DNA target sequences. It is calculated by the distance from the last hybridized nucleotide on the 5' end of the docking target site on the target strand to the first base on the 3' end of the mutation site.

[0055] In some embodiments, the docking array includes a secondary structure. In some embodiments, the docking array includes a stem-loop structure.

[0056] (Hairpin arrangement (ii))

[0057] The last nucleotide on the 3' end of the docking sequence is bound to the first nucleotide on the 5' end of the hairpin sequence (ii). The hairpin sequence does not need to be perfectly complementary to the sequence located immediately upstream of the docking target site of the mutant and wild-type DNA sequences, and does not need to hybridize with such sequences. The hairpin sequence (ii) hybridizes with the palm sequence (iv). In some embodiments, the hairpin sequence (ii) is perfectly complementary to the palm sequence (iv). In some embodiments, the hairpin sequence cannot hybridize with the mutant and wild-type DNA target sequences.

[0058] When the docking sequence hybridizes with the target sequence under specific annealing conditions (e.g., a specific annealing temperature) to stably position the switch primer on the common target DNA sequence (docking target site) according to the present invention, the hairpin sequence does not need to form a hybrid with the target sequence. Therefore, in some embodiments, the hairpin sequence does not hybridize with the target DNA sequence and / or bind to the target DNA sequence by base pairing at the annealing temperature of the docking sequence.

[0059] In some embodiments, the hairpin sequence according to the present invention is the same length as the palm sequence. In another embodiment, the hairpin sequence is about 1 to about 5 bases longer than the palm sequence. In yet another embodiment, the hairpin sequence is about 1 to about 3 bases shorter than the palm sequence.

[0060] In some embodiments, the hairpin sequence is about 50 to about 3 nucleotides long, preferably about 20 to about 3 nucleotides long, more preferably about 10 to about 4 nucleotides long, even more preferably about 8 to about 4 nucleotides long, and most preferably about 7 to about 5 nucleotides long.

[0061] (Spacer arrangement (iii))

[0062] The last nucleotide on the 3' end of the hairpin sequence is bound to the first nucleotide on the 5' end of the spacer sequence (iii). This spacer sequence acts as a spacer between the hairpin sequence and the palm sequence, enabling the formation of a two-dimensional hairpin structure (stem-loop) by the hairpin sequence, spacer sequence, and palm sequence. In some embodiments, the spacer sequence is not complementary to either the hairpin sequence (ii) or the palm sequence (iv). In some embodiments, the spacer sequence (iii) does not hybridize with the target DNA sequence at the annealing temperature of the docking sequence (i). In certain embodiments, the spacer sequence forms a hybrid with the target DNA sequence.

[0063] In some embodiments, the spacer sequence is about 20 to about 3 nucleotides long, preferably about 10 to about 4 nucleotides long, more preferably 8 to about 4 nucleotides long, and most preferably 5 to about 3 nucleotides long.

[0064] (Palme sequence (iv))

[0065] The last nucleotide on the 3' end of the spacer sequence is bound to the first nucleotide on the 5' end of the palm sequence (iv). The palm sequence consists of sequences complementary to the mutant and wild-type DNA target sequences (palm target sites). In some embodiments, the hairpin sequence is fully complementary to the palm target sites located immediately downstream of the mutant and wild-type DNA target sequences on the target strand. In some embodiments, the palm sequence is partially complementary to the hairpin sequence to the extent that hairpin formation is possible between the hairpin sequence, the spacer sequence, and the palm sequence.

[0066] The palm sequence is complementary to the hairpin sequence, thereby enabling the formation of a hybrid between the hairpin sequence and the palm sequence. In some embodiments, the hairpin sequence and the palm sequence are perfectly complementary to each other. In some embodiments, the hairpin sequence is the reverse complement of the palm sequence. In some embodiments, the palm sequence can form a hybrid with the hairpin sequence that is about 3 to about 30 nucleotides long, more preferably about 4 to about 20 nucleotides long, and most preferably about 4 to about 10 nucleotides long.

[0067] In some embodiments, the ΔG of the stem-loop structure, which includes a hairpin array, a spacer array, and a palm array, is about 0 to about -15, preferably about 0 to about -10, more preferably about 0 to about -8, and most preferably -2 to about -8.

[0068] Hairpin sequences and spacer sequences do not need to exhibit perfect complementarity to the internal regions of the target DNA sequence, but portions of the spacer and / or hairpin sequences may partially match the internal sequence.

[0069] The target DNA sequence and hairpin sequence located downstream of the mutation site compete with the palm sequence for complementarity. In some embodiments, the target DNA sequence located downstream of the mutation site is identical to the hairpin sequence.

[0070] (Mutation recognition sequence (v))

[0071] The last nucleotide at the 3' end of the palm sequence is bound to the first nucleotide at the 5' end of the mutation recognition sequence (v). The mutation recognition sequence contains or consists of 1 to 4 nucleotides. At least one of these nucleotides (mutation-specific nucleotide / interrogating nucleotide) is characterized by being a mismatch with each nucleotide at the mutation site of the wild-type DNA target sequence, while being complementary to the mutation site of the mutant DNA target sequence. In a preferred embodiment, the mutation recognition sequence consists of 2 nucleotides, and more preferably 1 nucleotide. In some embodiments, the mutation-specific nucleotide is the first or second nucleotide of the mutation recognition sequence. In a preferred embodiment, the mutation-specific nucleotide is the first nucleotide of the mutation recognition sequence.

[0072] To prevent the risk of the mutation recognition sequence being incorporated into the stem-loop formed by the hairpin sequence, spacer sequence, and palm sequence, and to enable optimal target binding, the docking sequence may further include a dimer-preventing nucleotide at the 3' end. The dimer-preventing nucleotide does not pair with the mutation-specific nucleotide and / or any other nucleotides in the 3' end and / or mutation recognition sequence in the multipart amplification primer. Therefore, in some embodiments, the mutation recognition sequence does not form part of the stem-loop structure. In some embodiments, the mutation recognition sequence is not part of the stem-loop structure. In some embodiments, the dimer-preventing nucleotide is the same nucleotide as the mutation-specific nucleotide. In some embodiments, the dimer-preventing nucleotide is roq nucleic acid (LNA). In some embodiments, the dimer-preventing nucleotide is ribonucleic acid. In some embodiments, the nearest nucleotide located upstream of the dimer-preventing nucleotide is ribonucleic acid.

[0073] While we do not wish to be bound by any theory, we hypothesize that mutation-specific nucleotides, which are complementary to the last base on the 3' end of the mutation site on the target strand of the mutant DNA target sequence, promote hybridization between the palm sequence and the mutant DNA target sequence at the palm target site of the mutant DNA target sequence. Because the mutation-specific nucleotides of the mutation-recognition sequence are mismatched with the mutation in the wild-type DNA target sequence, the palm target site of the wild-type DNA target sequence competes with the hairpin sequence for hybridization with the palm sequence, significantly reducing the stable formation of a hybrid between the palm sequence and the wild-type DNA target sequence.

[0074] This is further illustrated in Figure 2. Figure 2 shows embodiments of the switch primer described in the present invention and possible interactions with mutant and wild-type DNA target sequences, respectively. In the figure, the mutant DNA target sequence hybridizes with the palm sequence and mutation recognition sequence of the switch primer, while the wild-type DNA target sequence does not readily form such a hybrid because the mismatch of mutation-specific nucleotides in the mutation recognition sequence increases competition for hybridization between the hairpin sequence and palm sequence of the switch primer.

[0075] In some embodiments, the multipart amplification primer described in the present invention is a nucleotide sequence selected from the group consisting of polynucleotides described in SEQ ID NOs: 9-22, 32-35, 38, 39, 44, and 46-50. In some embodiments, the multipart amplification primer includes a nucleotide sequence selected from the group consisting of polynucleotides described in SEQ ID NOs: 9-22, 32-35, 38, 39, 44, 46-50, and a sequence having sequence identity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100%, or any range or value derived therefrom.

[0076] (Annealing temperature)

[0077] Annealing temperature during real-time PCR is a parameter that affects the specificity of target amplification. Generally, higher temperatures correlate with higher specificity of target amplification and lower off-target amplification. Such annealing temperatures for switch primers can be determined empirically by methods well known to those skilled in the art, such as systematically varying the annealing temperature or measuring it using gradient PCR that allows testing of multiple temperature conditions, although these methods are not limited to the following.

[0078] In some embodiments, the switch primer according to the present invention has an annealing temperature in a real-time PCR reaction, and the switch primer anneals to a target sequence contained in the sample at that temperature and dissociates at a temperature slightly higher than the annealing temperature of the amplification reaction. The annealing temperatures are approximately 40°C to 80°C, approximately 45°C to 80°C, approximately 50°C to 80°C, approximately 55°C to 80°C, approximately 56°C to 80°C, approximately 57°C to 80°C, approximately 58°C to 80°C, approximately 59°C to 80°C, and approximately 60°C to 80°C. In some embodiments, the annealing temperature is approximately 40°C to 70°C, approximately 40°C to 69°C, approximately 40°C to 68°C, approximately 40°C to 67°C, approximately 40°C to 66°C, approximately 40°C to 65°C, approximately 40°C to 64°C, approximately 40°C to 63°C, approximately 40°C to 62°C, approximately 40°C to 61°C, and approximately 40°C to 60°C, preferably approximately 50°C to 70°C, preferably approximately 55°C to 69°C, preferably approximately 55°C to 65°C, preferably approximately 56°C to 64°C, and preferably approximately 57°C to 63°C.

[0079] (Chimera switch primer)

[0080] Placing RNA bases within conventional DNA primers has been shown to reduce primer dimer formation and increase the efficiency and specificity of PCR reactions. While we do not wish to be bound by any theory or mechanism of action, it appears that nonspecific amplification products are generated mainly when base pairing occurs between the 3' end of a primer and a complementary base on another primer or probe, or when self-base pairing occurs between the 3' end of a primer and a complementary base on the same primer.

[0081] Surprisingly, it has been found that incorporating at least one ribonucleotide at a specific position in the switch primer can reduce or prevent nonspecific amplification products such as primer dimers, and further improve the efficiency of amplification of mutant DNA target sequences. Therefore, in some embodiments, multipart amplification primers include at least one ribonucleotide. In preferred embodiments, at least one ribonucleotide is located within 10 nucleotides from the 3' end of the primer, or within 10 nucleotides upstream from the expected start point of the primer dimer extension, with no two nucleotides adjacent to each other, and the 3' base is a deoxyribonucleotide.

[0082] In some embodiments, the switch primer is the sequence described in SEQ ID NOs. 33-35.

[0083] (Industrial applicability) The present invention further relates to a primer-dependent amplification and detection method capable of amplifying and detecting at least one mutant DNA target sequence in a sample. The method, utilizing switch primers according to the present invention, can amplify and detect at least one mutant DNA target sequence in a sample from a mixture containing closely related wild-type DNA target sequences that differ from the mutant DNA target sequence by only one or two base pairs. The method comprises steps (a) to (d).

[0084] The first step (a) of the method according to the present invention provides a sample containing at least one mutant DNA target sequence of which identification and / or detection is desired. Samples useful for the method according to the present invention may be prepared according to standard methods known to those skilled in the art (e.g., nucleic acid purification, reverse transcription, concentration, or a combination thereof). For example, the sample may be prepared from the biological / clinical sample of interest (e.g., whole blood sample, plasma sample, or tissue sample, but not limited to the following). In a preferred embodiment, the sample is prepared from a liquid biopsy. In some embodiments, the provided sample contains cfDNA isolated from a plasma sample.

[0085] In step (a), a pair of forward primers and reverse primers is further provided for each of at least one mutant DNA target sequence, wherein at least one of the forward primer and reverse primer is selective for the mutant DNA target sequence but mismatches with the wild-type DNA target sequence, characterized in that it is a multipart amplification primer (switch primer) according to the present invention. In a preferred embodiment, the forward primer is a switch primer.

[0086] In some embodiments, the reverse primer is a conventional primer. In some embodiments, the reverse primer is a multipart amplification primer according to the present invention. When both the forward primer and the reverse primer are multipart amplification primers, the forward primer has a mutation-specific nucleotide complementary to the mutation site in one of the two strands of the mutant DNA target sequence, and the reverse primer has a mutation-specific nucleotide complementary to the other mutant DNA sequence of its base pair in the other of the two strands of the mutant DNA sequence, so that one primer binds to the target strand and the other primer binds to the complementary target strand.

[0087] In the second step (b), the primer-dependent amplification mixture is prepared to include, or consist of, DNA polymerase, deoxyribonucleoside triphosphate, amplification buffer, other reagents necessary for amplification, the sample from step (a), and forward and reverse primer pairs for each mutant DNA target sequence from step (a).

[0088] In the next step (c), the mixture from step (b) undergoes repeated cycles of primer-dependent amplification under primer annealing conditions, including the primer annealing temperature, to amplify each mutant DNA target sequence present in the sample, thereby generating amplified products of the mutant DNA target sequences. In some embodiments, the docking sequences of the multipart amplification primers hybridize with the mutant DNA target sequences and closely related wild-type DNA target sequences at the primer annealing temperature of the docking sequences.

[0089] The use of amplification primers and the detection of primer-dependent amplification and target sequences are generally well known to those skilled in the art. A primer-dependent amplification reaction useful in the method according to the present invention may be any suitable exponential amplification method (including, but not limited to, polymerase chain reaction (PCR) (either symmetric or asymmetric PCR), ligase chain reaction (LCR), rolling circle amplification (RCA), etc.). In a preferred embodiment, the method according to the present invention utilizes PCR. The primer-dependent amplification reaction may include isothermal and / or repeated thermal cycles of primer annealing, primer extension, and chain denaturation.

[0090] Figure 3 shows an example of a primer-dependent amplification reaction for illustrative purposes. First, a multipart amplification forward primer (switched FWD primer, Figure 3I) anneals with the target strand of the mutant target sequence, generating the first extension product (forward amplicon) through polymerase-dependent synthesis (Figure 3II). Next, a second reverse primer (rev primer), similar to a conventional primer, binds to the forward amplicon and initiates the synthesis of the reverse amplicon (Figure 3III). In the next step, the forward primer hybridizes with the rev amplicon, initiating the synthesis of another further forward amplicon.

[0091] In the final step (d), at least one mutant DNA target sequence is detected by measuring the amount of the amplification product obtained in step (c). Detection may be performed after amplification by a method known to those skilled in the art, such as gel electrophoresis. Alternatively, homogeneous detection may be performed in a single tube, well, or other reaction vessel during the amplification reaction (real time) or after the amplification reaction (endpoint) using reagents included during amplification. The detection reagent includes double-stranded DNA bound to a staining reagent (e.g., SYBR Green), a fluorescently labeled hybridization probe capable of signal detection during hybridization (e.g., molecular beacon), a probe that cleaves during amplification (e.g., TaqMan probe), or any detection reagent known to those skilled in the art.

[0092] In some embodiments, the method enables the detection of at least one mutant DNA target sequence in a sample containing only one copy of at least one mutant DNA target sequence in a mixture containing 100 copies of closely related wild-type DNA target sequences that differ from the mutant DNA target sequence by only one or two base pairs. In preferred embodiments, the method enables the detection of at least one mutant DNA target sequence in a sample containing only 10 copies of at least one mutant DNA target sequence in a mixture containing 1,000 copies, more preferably 10,000 copies, of closely related wild-type DNA target sequences that differ from the mutant DNA target sequence by only one or two base pairs.

[0093] In diagnostic testing, simultaneously amplifying multiple target sequences enables cost-effective and rapid testing for various biomarkers, such as mutations in target sequences, as well as genotyping. In some cases, such as during RT-PCR, simultaneously amplifying multiple targets is necessary to measure the quantity of targets in a single reaction. Therefore, in amplification and / or detection assays, the ability of the switch primers according to the present invention to effectively amplify targets without interference from other primer pairs (background primers), unlike conventional primers, is beneficial in such assays.

[0094] Surprisingly, it has been found that the switch primers according to the present invention can selectively amplify the mutant target DNA sequences according to the present invention in the presence of multiple background primers. Therefore, in some embodiments of the method according to the present invention, for each mutant target DNA sequence, the method can amplify and / or detect at least one mutant target DNA sequence in a sample in a mixture containing closely related wild-type target DNA sequences that differ from the mutant target DNA sequence by only one or two base pairs, the sample further comprising at least one set of background primers, more preferably about 1 to about 30, about 1 to about 20, about 1 to about 15, about 5 to about 15, about 5 to about 10, and most preferably about 1 to about 20 sets of background primers.

[0095] (Switch primer design)

[0096] The design of switch primers according to the present invention may require optimization depending on the selected target DNA sequence. As is known in the art, primer design requires careful selection of sequences and lengths that can optimally amplify the intended target sequence. For example, adjusting the primer length to match the amplification protocol and the instrument used for amplification and / or detection is important to ensure successful amplification of the target sequence and reliability of detection.

[0097] Switch primer design may require adjustment of the length and sequence of five consecutive DNA sequences (i) to (v) containing the primer according to the present invention. For example, shortening or lengthening the docking sequence to achieve a desired melting temperature may allow the docking sequence to optimally bind to the target DNA sequence, thus further stabilizing the amplification of the mutant target DNA sequence. Furthermore, as is known to those skilled in the art, adjusting the placement of the docking sequence by sliding a window of a desired length on the target DNA sequence may improve binding characteristics. Similarly, adjusting the structure of the hairpin formed by the hairpin sequence, spacer sequence, and palm sequence may allow for proper cleavage of the hairpin when binding to each mutant target DNA sequence containing the mutation site, potentially affecting optimal target binding during amplification.

[0098] Those skilled in the art will know the steps required to optimize primer sequences for the design of conventional primers and more advanced primers (e.g., multipart primers). First, it is proposed to select a switch primer design for any desired target DNA sequence having sequences (i) to (v) of specified lengths, with the mutation-specific nucleotide positioned at the first or second nucleotide of the mutation-recognition sequence (v). Next, it is recommended to incorporate a ribonucleotide into the switch sequence at the position described herein, comprising the dimer-preventing nucleotide according to the present invention. After optimizing the primer as described above, it may be useful to test the switch sequence and amplification / detection protocol using a sample containing known concentrations of the mutant target DNA sequence and the wild-type DNA sequence. In this case, by performing multiple trials with variations in several properties, such as the length and sequence of parts (i) to (v) of the switch primer according to the present invention, sufficient specificity and selectivity can be ensured to detect extremely low copy number mutant target DNA sequences in samples containing high concentrations of the wild-type target DNA sequence. Several protocols and optimization methods are shown in the Examples section. However, it should be noted that the suggestions and examples for primer design optimization are for illustrative purposes only and not limiting purposes.

[0099] (kit)

[0100] A reagent kit for carrying out the method according to the present invention is further disclosed.

[0101] The kit may include one or more pairs of multipart amplification primers according to the present invention for one or more mutant DNA target sequences required for amplification, dNTPs, primer-dependent polymerases, detection probes for each mutant DNA target, and other reagents (in particular amplification buffers). One of the primers in each primer pair may be a multipart amplification primer, and the other primer in each primer pair may be a conventional primer or a multipart amplification primer.

[0102] The kit may be used in any application involving nucleic acid amplification reactions, such as diagnostic kits for cancer screening.

[0103] In some embodiments, the kit may further include a detection reagent capable of detecting mutant DNA target sequences. [Brief explanation of the drawing]

[0104] [Figure 1] Figure 1 shows a schematic diagram of one embodiment of a switch primer according to the present invention. Sequences (i) to (v), which are the continuous DNA sequences of the primer, are shown. [Figure 2] Figure 2, Panel A shows schematic diagrams of mutant and wild-type DNA target sequences according to the present invention. Panel B shows schematic diagrams of possible binding of the switch primer to either the mutant or wild-type DNA target sequence. [Figure 3] Figure 3 shows a schematic diagram of the amplification cycle and detection of the amplicon according to the method of the present invention. [Figure 4] Figure 4 shows the secondary structure of an embodiment illustrating the switch primer design according to the present invention (SEQ ID NO: 44). The bases, 5' and 3' ends, and base numbers counted from the 5' end are shown. Bases 1-26 correspond to the docking sequence (i) described in the present invention, bases 27-31 correspond to the hairpin sequence (ii), bases 32-35 correspond to the spacer sequence (iii), bases 36-40 correspond to the palm sequence (iv), and base 41 corresponds to the mutation recognition sequence (v). [Figure 5] Figure 5 shows the interaction between the switch primer (right strand, SEQ ID NO: 44) from Figure 4 and its mutant DNA target sequence (left strand, SEQ ID NO: 45). Counting from the first base at the 5' end of the mutant DNA target sequence, the first base corresponds to the mutation site, bases 2-6 correspond to the palm target site, bases 7-23 correspond to the internal site, and bases 24-47 correspond to the docking target sequence. [Figure 6]Figure 6 illustrates a specific embodiment of the present invention, in which the switch primers of Figures 4 and 5 incorporate ribonucleotides (circled bases) at positions 26 and 40 (SEQ ID NO: 46). [Figure 7] Figure 7 shows the real-time qPCR results analysis of the PCR assay described in Example 1. [Figure 8] Figure 8 shows the real-time qPCR results analysis of the PCR assay described in Example 2. [Figure 9] Figure 9 shows the real-time qPCR results analysis of the PCR assay described in Example 3. [Figure 10] Figure 10 shows the real-time qPCR results analysis of the PCR assay described in Example 4. [Figure 11] Figure 11 shows the real-time qPCR results analysis of the PCR assay described in Example 5. [Figure 12] Figure 12 illustrates the multipart amplification design, in which the mutation-specific nucleotide is incorporated into a hairpin structure (SW-1, SEQ ID NO: 47). [Figure 13] Figure 13 illustrates the multipart amplification design according to the present invention, where the mutation-specific nucleotide is located at the 3' end and is not part of the hairpin (SW-2, SEQ ID NO: 48). [Figure 14] Figure 14 illustrates the multipart amplification design according to the present invention, where the mutation-specific nucleotide is located at the 3' end and is not part of the hairpin. Furthermore, the palm sequence is partially complementary to the hairpin sequence to the extent that hairpin formation is possible between the hairpin sequence, spacer sequence, and palm sequence, and as a result, unpaired nucleotides can form a bubble structure within the hairpin structure (SW-3, SEQ ID NO: 49). [Figure 15]Figure 15 illustrates a multipart amplification design, where the mutation-specific nucleotide is located at the 3' end of the primer, and an additional nucleotide is placed between the palm sequence and the mutation-specific nucleotide, neither of which is part of the hairpin structure (SW-4, SEQ ID NO: 50). [Figure 16] Figure 16 illustrates a multipart amplification design consisting of a hairpin sequence, where the mutation-specific nucleotide is located at the 3' end of the primer, and an additional nucleotide is positioned between the palm sequence and the mutation-specific nucleotide, neither of which is part of the hairpin structure (SL-1, SEQ ID NO: 51). [Figure 17] Figure 17 illustrates a multipart amplification design consisting of a hairpin sequence, where the mutation-specific nucleotide is located at the 3' end of the primer, and two additional nucleotides are positioned between the palm sequence and the mutation-specific nucleotide; these three nucleotides are not part of the hairpin structure (SL-2, SEQ ID NO: 52). [Figure 18] Figure 18 illustrates a multipart amplification design consisting of a hairpin sequence, where the mutation-specific nucleotide is located at the 3' end of the primer, and three additional nucleotides are positioned between the palm sequence and the mutation-specific nucleotide, with these four nucleotides not being part of the hairpin structure (SL-3, SEQ ID NO: 53). [Figure 19] Figure 19 illustrates a multipart amplification design consisting of a hairpin sequence, where the mutation-specific nucleotide is located at the 3' end of the primer, and four additional nucleotides are positioned between the palm sequence and the mutation-specific nucleotide, with these five nucleotides not being part of the hairpin structure (SL-3, SEQ ID NO: 54). [Figure 20] Figure 20 shows the real-time qPCR results analysis of the PCR assay described in Example 6. [Examples]

[0105] (Example 1) (Switch primers selectively amplify mutant target DNA sequences.) To explain the higher selectivity and specificity for amplification of mutant DNA target sequences compared to wild-type DNA target sequences, real-time qPCR experiments were devised.

[0106] (DNA template)

[0107] Three different DNA templates were used in real-time PCR experiments. The first DNA template, named "Mut gB," was a synthetic gBlock containing a 250-nucleotide sequence (mutant DNA target sequence) with the mutation of interest. Mut gB was synthesized by IDT DNA Inc., diluted to 4.15 nM, and further diluted as directed for each experiment. The second DNA template, named "WT," was extracted from the glioblastoma U87 cell line and contained the wild-type DNA target sequence. Genomic DNA was diluted 340 ng / 1 μl of stock solution and further diluted as directed. The wild-type and mutant DNA target sequences differed by one mutation (mutation site). The third DNA template was a mixture of Mut gB and WT DNA in varying proportions, allowing for evaluation of the sensitivity and specificity of the PCR assay for detecting the mutation of interest. Positive control primers, named "Region" primers, were used to determine the copy number ratios of the Mut gB and WT DNA templates. The "Region" primer targeted sequences adjacent to the test mutation and served as a positive control and quantitative standardization for the PCR assay.

[0108] (Primers and probes)

[0109] Switch primers were designed to target the following specified human gene mutations (mutation target DNA sequences): KRAS G12D (SEQ ID NO: 1), EGFR L858R (SEQ ID NO: 3), CTNNB1 S33C (SEQ ID NO: 2), and SMAD4 R361H (SEQ ID NO: 4). The wild-type DNA sequence differs from the mutant DNA sequence by one mutation (SNP), and the wild-type target DNA sequences are KRAS target SEQ ID NO: 5, EGFR target SEQ ID NO: 6, CTNNB1 target SEQ ID NO: 7, and SMAD4 target SEQ ID NO: 8. Two types of experiments were performed: singleplex and multiplex experiments. In the singleplex experiment, each reaction tube consisted of a single primer set. In the multiplex experiment, each reaction tube consisted of a test primer set and an additional group of 20 primer sets targeting different cancer mutations, which served as background. For the singleplex primer mix, the primers were prepared by diluting them in 4 mM stock solution and then further diluting them to a final concentration of 200 nM in the reaction tube. Each reactant contained a primer set consisting of forward-switch primers and standard DNA reverse primers at the same concentration. For the multiplex primer mix, each primer was diluted in 100 μM stock solution. The forward and reverse primers were then mixed into two separate primer mixes, one for the target and one for the background, respectively. Each primer mix was prepared in the reaction tube to a final concentration of 4 μM for the primer mix and 200 nM for each primer.

[0110] Multiplex experiments were performed using TaqMan probes double-labeled with 5' Fam and Black Hole Quencher 1. The probes were diluted with target primers, with final concentrations set at 3 μM in the target primer mix and 150 nM in the reaction tube.

[0111] In addition to standard switch DNA primers, switch chimeric primers (named Switch-Chi) targeting SMAD4 R361H were also designed. In the switch chimeric primers, two positions were modified with RNA bases. The positions of the RNA chimeric bases were selected from locations presumed to be the start sites of DNA polymerase elongation. Specifically, the first site was placed near a site suspected of self-elongation, and the second site was placed closest to the mutation site at the 3' end.

[0112] All primers and probes were synthesized by IDT DNA Inc.

[0113] (Real-time PCR settings)

[0114] Real-time PCR enzyme mixture is ORA for SyBR green reaction (商標) SEE qPCR Green ROX L Mix (HighQu GmbH, Germany), or ORA for TaqMan probe reactions (商標) One of the following was used: SEE qPCR Probe Mix (HighQu GmbH, Germany). [ka] [ka]

[0115] All real-time PCR experiments were performed using an Opus CFX 96 (Bio-Rad, USA) with the following thermocycler programs shown in Table 1. [Table 1]

[0116] In the TaqMan protocol, the melting curve protocol (step number 10 in the program) is omitted, and the fluorophore has been replaced from SYBR green to FAM or HEX.

[0117] (Real-time PCR data analysis)

[0118] The sensitivity value was calculated using the formula: Cq[NTC]-Cq[target], where NTC is the negative template control.

[0119] The specificity value was calculated using the formula: Cq[WT]-Cq[mut].

[0120] If amplification did not occur and a Cq value could not be obtained, a fixed value of Cq=38 was assigned. This value was determined by calculating the average of the normally distributed Cq values ​​obtained from all negative template control (NTC) reactions performed beforehand.

[0121] (result)

[0122] First, to determine the best design, several sets of switch primers for cancer mutations were designed. The switch-forward primers that specifically target the mutant target DNA sequence of KRAS were KRAS_Switch 1, 2, 4, and 5 (sequence numbers 9, 10, 11, and 12, respectively), for CTNNB1 were CTNNB1_Switch 1, 3, 4, and 6 (sequence numbers 13, 14, 15, and 16, respectively), and for EGFR were EGFR_Switch 1 nat, 2 nat, 4, 5, 8, and 9 (sequence numbers 17, 18, 19, 20, 21, and 22, respectively). The reverse primers for each primer set used were the same for all region-specific sets. For KRAS-specific amplification (Figure 7A), the KRAS_rev primer (sequence number 23) was used; for CTNNB1-specific amplification (Figure 7B), the CTNNB1_rev primer (sequence number 24) was used; and for EGFR-specific amplification (Figure 7C), the EGFR_rev primer (sequence number 25) was used. A positive control was included, and the forward primer was a conventional primer that did not distinguish between mutant and wild-type target DNA sequences (Region, positive control). For KRAS-specific amplification (Figure 7A), the KRAS_fwd primer (SEQ ID NO: 26) was used; for CTNNB1-specific amplification (Figure 7B), the CTNNB1_fwd primer (SEQ ID NO: 27) was used; and for EGFR-specific amplification (Figure 7C), the EGFR_fwd primer (SEQ ID NO: 28) was used. The reference reaction was performed using a state-of-the-art primer design capable of distinguishing between mutant and wild-type sequences (SuS-primer strategy, Kramer FR, Vargas DY. SuperSelective primer pairs for sensitive detection of rare somatic mutations. Sci Rep. 2021 Nov 17;11(1):22384. doi: 10.1038 / s41598-021-00920-4. PMID: 34789731; PMCID: PMC8599793).For SuS forward primers, the KRAS_SuS primer (SEQ ID NO: 29) was used for KRAS-specific amplification (Figure 7A), the CTNNB1_SuS primer (SEQ ID NO: 30) for CTNNB1-specific amplification (Figure 7B), and the EGFR_SuS primer (SEQ ID NO: 31) for EGFR-specific amplification (Figure 7C). The charts shown in Figures 7A-C illustrate the specificity of the switch primers in singleplex experiments targeting KRAS G12D, CTNNB1 S33C, and EGFR L858R (mutant DNA target sequences) in samples containing both mutant and wild-type target DNA sequences. For each mutation, most of the switch primers showed superior performance compared to previously published superselective (Sus) primers. Some primers showed 1000-fold higher specificity to the mutant sequence than to the wild type (ΔCq 9-11), while others showed more than 10,000-fold higher specificity to the mutant (ΔCq > 13).

[0123] (Example 2) (Highly specific amplification of mutant target DNA sequences) Identifying mutant target sequences in samples containing both low-copy-number mutant and high-copy-number wild-type DNA target sequences is essential for ctDNA identification. Multiple dilutions of mutant and wild-type sequences were prepared to demonstrate that switch primers can specifically identify low-copy-number mutant DNA target sequences while being rich in corresponding wild-type DNA target sequences. RT-PCR assays were performed and analyzed as described in Example 1.

[0124] In more detail, the sensitivity of switch primer set 1 targeting the KRAS G12D mutation was tested by serially diluting the mutant and wild-type templates together until the mutant:wild-type ratio reached 1:1000.

[0125] The results are summarized in Figure 8. Adding wild-type genomic DNA did not significantly affect the specificity of KRAS switch 1 for the KRAS G12D mutation. Specificity changed from ΔCq 15.59 (equivalent to more than 10,000 times) when the ratio of mutant gBlocks was 1:1000, to ΔCq 25.56 when the ratio was 1:1.

[0126] (Example 3) (Switch primers efficiently amplify the target sequence in the presence of background primers.) In some cases, simultaneously amplifying multiple targets during RT-PCR is necessary to measure the number of targets in a single reactant. Therefore, it is beneficial in such assays for primers to efficiently amplify targets without interference from other primer pairs (background primers). The RT-PCR assay was performed and analyzed as described in Example 1.

[0127] Therefore, in the following experiment, a multiplex assay was performed to test the performance of switch primer set 1 targeting KRAS G12D by serially diluting mutant / wild-type templates in the presence of 20 background primers.

[0128] The switch primers, containing a set of 20 different primers in a tube, showed high specificity under conditions where mutant:wild-type molecules were present in a 1:100 ratio.

[0129] The results are shown in Figure 9.

[0130] (Example 4) (Chimera switch primer) In the following experiments, a switch-forward primer (SMAD4_Switch 3, SEQ ID NO: 32) that specifically amplifies the SAMD4 mutant target DNA sequence was modified by changing 1-2 base positions from DNA to RNA to prepare a switch-chimeric primer. The first modification site was closest to the 3' end of the primer (SMAD4_Switch chi 3.1, SEQ ID NO: 33). The second modification site was located in the center of the primer, near the expected site of the primer's self-extension (SMAD4_Switch chi 3.2, SEQ ID NO: 34). In addition, a third primer with both positions modified was designed (SMAD4_Switch chi 3.3, SEQ ID NO: 35). The switch primers targeted the SMAD4 R361H mutant and were tested under singleplex conditions. A conventional forward primer was used as a positive control to amplify both the mutant and wild-type target DNA sequences (SMAD4_region_fwd, SEQ ID NO: 36). The reverse primer for all reactions was SMAD4_rev (SEQ ID NO: 37). The RT-PCR assay was performed and analyzed as described in Example 1.

[0131] Modification from DNA to RNA (chi 3.1) closest to the 3' end base of the primer increases specificity by 3.5 times (1.84 cycles). RNA modification in the center of the primer, close to the self-extension site and the predicted site (chi 3.2), increases specificity by 36 times (5.16 cycles). RNA modification at both locations (chi 3.3) increases specificity by 150 times (7.29 cycles).

[0132] The results show that modifying the switch primer at both locations increases specificity compared to modifying the RNA at only one location.

[0133] The results are shown in Figure 10. As shown, incorporating ribonucleotides into the switch primer significantly increases specificity.

[0134] (Example 5) (Mutation-specific nucleotide arrangement) In some embodiments of the present invention, the mutation-specific nucleotide included in the mutation recognition sequence of the switch primer may be positioned at the second position of the sequence. Two switch designs were investigated to demonstrate the effect of the interrogating base position on specificity. Switch primer C420R switch 2 (SEQ ID NO: 38) contains the mutation-specific nucleotide at the first position of the mutation recognition sequence. On the other hand, switch primer 3'2nd position (SEQ ID NO: 39) contains the mutation-specific nucleotide at the second position of the mutation recognition sequence. Both sequences were designed for PIK3CA-specific mutations (PI3KCA mutant DNA target sequence SEQ ID NO: 40, PI3KCA wild-type DNA target sequence SEQ ID NO: 41). The reverse primer for all reactions was SEQ ID NO: 42, and the positive control (Region control, conventional fwd primer) was SEQ ID NO: 43. RT-PCR assays were performed and analyzed as described in Example 1.

[0135] Switch 2, targeting the PIK3CA C420R mutation, showed high specificity with a △△Cq value of 13.05 for 1 / 10000 mut / wt. A primer with the mutant complementary base at the second position upstream of the 3' end showed a △△Cq value of 10.2. This ratio is greater than 1 / 1000 mut / wt. This ratio is sufficient for ctDNA samples. Based on these results, the position of the mutant complementary base may also be the second position from the 3' end of the primer. The results are shown in Figure 11.

[0136] (Example 6) (Evaluation of primer design) To evaluate the effects and interactions of various parts of the multipart amplification primers of the present invention on the selective amplification of target DNA sequences rather than closely related DNA sequences, further qRT-PCR experiments were devised, and cleaved and modified primer designs, for example, known to those skilled in the art, were tested and compared. The primer set was designed to target EGFR L858M (SEQ ID NO: 55). The reverse primer of each primer set used is identical for all region-specific sets. For EGFR-specific amplification, the EGFR_rev primer (SEQ ID NO: 25) was used.

[0137] The experiment was carried out using primer pairs SW1, SW2, SW3, SW4, SL1, SL2, SL3, and SL4, as previously described in Example 1, and after amplifying the target DNA sequence, the average ΔCq value was calculated for each primer pair. SW1 (Figure 12, Sequence ID 47) demonstrates a multipart amplification design, where mutation-specific nucleotides are incorporated into a hairpin structure; SW2 (Figure 13, Sequence ID 48) illustrates the multipart amplification design according to the present invention, where the mutation-specific nucleotide is located at the 3' end and is not part of the hairpin; SW3 (Figure 14, Sequence ID 49) illustrates the multipart amplification design according to the invention, where the mutation-specific nucleotide is located at the 3' end, and the palm sequence is partially complementary to the hairpin sequence to the extent that hairpin formation is possible between the hairpin sequence, spacer sequence, and palm sequence, rather than being part of the hairpin; as a result, unpaired nucleotides can form a bubble structure within the hairpin structure; SW4 (Figure 15, Sequence ID 50) shows a multipart amplification design, where the mutation-specific nucleotide is located at the 3' end of the primer, and an additional nucleotide is placed between the palm sequence and the mutation-specific nucleotide, neither of which is part of the hairpin structure; SL1 (Figure 16, Sequence ID 51) exhibits a multipart amplification design consisting of a hairpin sequence, with the mutation-specific nucleotide located at the 3' end of the primer, and an additional nucleotide placed between the palm sequence and the mutation-specific nucleotide, neither of which is part of the hairpin structure; SL2 (Figure 17, Sequence ID 52) shows a multipart amplification design consisting of a hairpin sequence, with the mutation-specific nucleotide located at the 3' end of the primer, and two additional nucleotides placed between the palm sequence and the mutation-specific nucleotide, the three nucleotides not being part of the hairpin structure; SL3 (Figure 18, Sequence ID 53) exhibits a multipart amplification design consisting of a hairpin sequence, with the mutation-specific nucleotide located at the 3' end of the primer, and three additional nucleotides placed between the palm sequence and the mutation-specific nucleotide, the four nucleotides of which are not part of the hairpin structure; SL4 (Figure 19, Sequence ID 54) exhibits a multipart amplification design consisting of a hairpin sequence, with the mutation-specific nucleotide located at the 3' end of the primer, and four additional nucleotides placed between the palm sequence and the mutation-specific nucleotide, the five nucleotides of which are not part of the hairpin structure.

[0138] The chart in Figure 20 shows the specificity of various primer designs in singleplex experiments targeting EGFR L858M (the mutant DNA target sequence) in a sample containing both mutant and wild-type target DNA sequences. Interestingly, there were significant differences in the selective amplification of the target sequence depending on the design used. Designs lacking the docking sequence (SL1-SL4) generally failed to achieve specificity greater than ~150 (△Cq<7.2). On the other hand, it was suggested that the placement of the mutant-specific nucleotide itself makes a further contribution, and it was shown that the optimal placement was when the nucleotide was located at the 3' end of the primer and directly downstream of the hairpin structure. Adding further nucleotides between the hairpin and the mutant-specific nucleotide significantly reduced specificity. Similar effects were measured for primer designs incorporating the docking sequence according to the present invention (SW1-SW4). In this case as well, placing the mutant-specific nucleotide directly downstream of the hairpin structure yielded the highest specificity (SW2 and SW3>10,000).

[0139] In summary, the above results demonstrate a clear relationship between the specificity of target DNA amplification and the presence of the docking sequence and the arrangement of mutation-specific nucleotides according to the present invention.

Claims

1. A multipart amplification primer that distinguishes between a mutant DNA target sequence and a closely related wild-type DNA target sequence, wherein the following is observed in the 5' to 3' direction: (i) A docking sequence that can form a hybrid of 15 to 40 nucleotides in length with a mutant DNA target sequence and a wild-type DNA target sequence. (ii) Hairpin sequences that cannot form hybrids with mutant DNA target sequences and wild-type DNA target sequences, (iii) Spacer arrangement, (iv) Palm sequences that are complementary to the mutant DNA target sequence and the wild-type DNA target sequence and can form a 4-10 nucleotide hybrid with the hairpin sequence, and (v) A mutation recognition sequence containing the first or second mutation-specific nucleotide located at the 5' end of the sequence. It is characterized by consisting of, or containing, five consecutive DNA sequences. The continuous hairpin arrangement, spacer arrangement, and palm arrangement form a stem-loop structure. The mutant target DNA sequence is completely complementary to the sequence formed by the palm sequence and the mutation recognition sequence. The wild-type DNA target sequence has a mismatch with the sequence containing the mutation-specific nucleotide of the mutant recognition sequence, and A multipart amplification primer wherein the docking sequence further contains a dimer-preventing nucleotide at the last position of the 3' end, which does not pair with the mutation-specific nucleotide and / or any other nucleotide located at the 3' end of the multipart amplification primer.

2. The dimer-preventing nucleotide is identical to the nucleotide located at the beginning of the 5' end of the mutation recognition sequence. The multipart amplification primer according to claim 1.

3. The dimer-preventing nucleotide in question is a lock nucleic acid (LNA). A multipart amplification primer according to claim 1 or 2.

4. Furthermore, it contains at least one ribonucleotide, A multipart amplification primer according to any one of claims 1 to 3.

5. At least one ribonucleotide is located within 10 nucleotides from the 3' end of the primer, the two ribonucleotides are not adjacent to each other, and the 3' terminal base is a deoxyribonucleotide. The multipart amplification primer according to claim 4.

6. A nucleotide sequence selected from the group of polynucleotides described in SEQ ID NOs: 9-22, 32-35, 38, 39, 44, and 46-50, or consisting of such a sequence. A multipart amplification primer according to any one of claims 1 to 4.

7. A primer-dependent amplification and detection method is provided, which allows for the amplification and detection of at least one mutant DNA target sequence in a sample within a mixture containing a closely related wild-type DNA target sequence that differs from the mutant DNA target sequence by only one or two base pairs, comprising the following steps: (a) To provide a sample containing at least one mutant and / or wild-type DNA target sequence, and for each mutant DNA target sequence, to provide a pair of forward primers and reverse primers. (b) Prepare a primer-dependent amplification reaction reaction comprising, or consisting of, DNA polymerase, deoxyribonucleoside triphosphate, amplification buffer, other reagents necessary for amplification, the sample, and forward and reverse primer pairs for each mutant DNA target sequence of step (a). (c) Repeatedly cycle the primer-dependent amplification reaction on the mixture obtained therefrom under primer annealing conditions including the primer annealing temperature, to amplify each mutant DNA target sequence contained in the sample, and (d) Detecting at least one mutant DNA target sequence by measuring the amount of the amplification product obtained thereby. This includes, or substantially consists of; A multipart amplification primer according to any one of the claims, characterized in that the forward primer and / or reverse primer of the primer pair for each mutant DNA target sequence are specific to the mutant DNA target sequence but have a mismatch with respect to the wild-type DNA target sequence, A method comprising hybridizing the docking sequence with a mutant DNA target sequence and a closely related wild-type DNA target sequence at the primer annealing temperature.

8. For each variant DNA target sequence, either the forward primer or the reverse primer of the primer pair is a conventional primer. The method according to claim 7.

9. If the docking sequence and palm sequence of the primer hybridize with either a mutant DNA target sequence or a wild-type DNA target sequence, the probability that the multipart amplification primer / wild-type DNA target sequence hybrid will be extended during that cycle is at least 1,000 times lower than the probability that the multipart amplification primer / mutant DNA target sequence hybrid will be extended during that cycle. This is supported by a difference in the number of thermal cycles (ΔCq) of at least 10. The method according to claim 7 or 8.

10. The forward primer and reverse primer of the forward primer and reverse primer pair are the multipart amplification primers described in any one of claims 1 to 6. The forward primer has a mutation-specific nucleotide that is complementary to the mutation site in one of the two strands of the mutant DNA target sequence. The reverse primer has a mutation-specific nucleotide that is complementary to the other mutant DNA target sequence in the other strand of the double helix of the mutant DNA target sequence, thereby allowing one primer to bind to the target strand. The other primer binds to its complementary target chain. The method according to any one of claims 7 to 9.

11. The closely related wild-type DNA target sequence differs from the mutant DNA target sequence by one base. The method according to any one of claims 7 to 10.

12. The docking sequence hybridizes with mutant and wild-type DNA target sequences at the primer annealing temperature. The method according to any one of claims 7 to 11.

13. The primer-dependent amplification mixture of step (b) further comprises homogeneous fluorescence detection means for detecting the amplification product, and Step (d) includes detecting at least one mutant DNA target sequence by measuring the fluorescence intensity emitted from the homogeneous fluorescence detection means. The method according to any one of claims 7 to 12.

14. The primer-dependent amplification and detection method is polymerase chain reaction (PCR), and the detection is real-time (RT) detection. The method according to claim 13.

15. For each mutant DNA target sequence, a mixture containing 10,000 copies of closely related wild-type DNA target sequences that differ from the mutant DNA target sequence by only one or two base pairs can amplify and detect at least one mutant DNA target sequence, of which only 10 copies exist, in the sample. The method according to any one of claims 7 to 14.

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