Nucleic acid amplification method and application thereof in sequencing

Abstract

The invention provides a nucleic acid amplification method, a single-stranded DNA multi-copy catenary and application thereof. The method comprises the following steps: connecting nucleic acid with a known sequence to obtain nucleic acid to be amplified; designing a primer pair according to a known sequence contained in nucleic acid to be amplified, and carrying out amplification ligation reaction to obtain a first double-stranded DNA multi-copy catenary; two primers of the primer pair can respectively contain different restriction enzyme cutting sites 1 and 2. An enzyme 1 can be used for cutting an enzyme cutting site 1 contained in the double-stranded DNA multi-copy catenary, a single-stranded gap is formed in a double-stranded DNA ring, and a digestive enzyme is used for digesting the single-stranded gap so as to obtain a first single-stranded DNA multi-copy catenary. Taking the first single-stranded DNA multi-copy catenary as a template, and carrying out amplification ligation reaction by utilizing the primer to obtain a second double-stranded DNA multi-copy catenary; and cutting an enzyme cutting site 2 contained in the second double-stranded DNA multi-copy catenary by using an enzyme 2, forming a notched single strand in the second double-stranded DNA multi-copy catenary, and digesting the notched single strand by using a digestive enzyme so as to obtain the second single-stranded DNA multi-copy catenary. The single-stranded DNA multi-copy catenary is used for nucleic acid sequencing.

Description

A nucleic acid amplification method and its use in sequencing Technical Field

[0001] The present disclosure relates to the field of molecular biology technology, specifically, the field of nucleic acid amplification, and more specifically, to a nucleic acid amplification method, a single-stranded DNA multi-copy concatemer, and uses thereof. Background Art

[0002] In the field of life science research, sequencing technology has become one of the most commonly used and important research tools. Since Sanger's invention of the dideoxy chain termination method in 1977, the first generation of sequencing technology, and the impetus of the Human Genome Project, first-generation sequencing technology has evolved from isotope labeling and flat-plate electrophoresis to fluorescent labeling, capillary electrophoresis, and automated imaging systems, significantly improving sequencing speed and throughput. However, compared to the growing demand for sequencing, the advanced first-generation sequencing technology still cannot meet the demand. This is why next-generation sequencing technology was invented and has rapidly developed in a short period of time. Next-generation sequencing technology, also known as second-generation sequencing technology or massively parallel sequencing technology, is characterized by its ability to sequence large numbers of DNA fragments (millions to billions) simultaneously. Compared to first-generation Sanger sequencing, it has significantly improved sequencing speed and throughput, further meeting the sequencing needs of scientific research and applications. The typical steps in massively parallel sequencing technology involve fragmenting DNA and adding adapters of known sequence to form a library. These adapter-added DNA fragments are then loaded onto a sequencing chip. Each adapter-added DNA fragment in the library occupies a specific position on the chip, allowing all sequences on the chip to be sequenced simultaneously, thus achieving the goal of massively parallel sequencing. This process includes a crucial step: sequencing template signal amplification. This is because a single copy of a DNA sequence, whether relying on fluorescence or ion signals, does not provide sufficient information intensity. Therefore, before loading the DNA sequence onto the chip, each single copy of the DNA fragment in the library must be amplified. This amplification process amplifies the single copy of the DNA sequence into multiple copies of a monoclonal DNA sequence at a fixed spatial location, generating so-called base signal acquisition units. This increases signal intensity and ensures that the signal can be distinguished from background noise. It also facilitates the formation of independent reaction centers, allowing all reaction centers to react simultaneously, collect information, perform signal detection and interpretation, and thus sequentially read the sequence information of the DNA fragments. The number of copies of the amplified DNA fragment and the resulting space occupied by these multiple copies of the template significantly impact sequencing accuracy, read length, and throughput. A greater number of copies results in a stronger signal, a higher signal-to-noise ratio, and improved sequencing accuracy. A greater number of copies mitigates asynchrony between copies of the same type during sequencing, improving read length. Ultimately, the smaller the space occupied by these multiple copies of the template, the more reaction centers can be arranged on the limited sequencing chip, potentially increasing sequencing throughput. Therefore, template amplification is one of the most critical core technologies for massively parallel sequencing.

[0003] Since the invention of large-scale sequencing technology, the technologies for amplifying sequencing DNA templates to form so-called base signal acquisition units can be mainly divided into three types: microbead emulsion PCR amplification, solid-phase bridge PCR amplification, and DNA nanoball amplification.

[0004] Bead-emulsion PCR amplification is primarily used on Roche's 454 sequencing platform and Thermo Fisher's SOLiD and Ion torrent platforms. The specific steps involve mixing a DNA library constructed with adapter sequences with microbeads bearing sequences complementary to the adapters, dNTPs, primers, and DNA polymerase. A specific mineral oil and surfactant are then added, and the reaction system is vigorously shaken to form a stable water-in-oil emulsion. Under ideal conditions, each droplet will contain only a single magnetic bead and a single strand of single-stranded DNA. By controlling the conditions of this step, at least 10^6 ideal droplets can be formed in 1 mL of emulsion. After PCR amplification, each bead surface is covered with thousands of copies of the same DNA sequence. The emulsion mixture is then broken, leaving the amplified fragments still bound to the magnetic beads.

[0005] Solid-phase bridge PCR amplification is primarily used on various Illumina sequencing platforms. In solid-phase bridge PCR amplification, fragmented DNA is ligated with adapters to form a library. This adapter-containing library is converted to single strands, diluted to an appropriate concentration, and added to a sequencing chip. The single-stranded DNA fragments bind to the sequencing chip via complementary adapters. Because the library concentration after dilution is sufficiently low, it is assumed that the library fragments are evenly bound to the chip surface, with the binding sites of each fragment sufficiently far apart. The single-stranded DNA that hybridizes with the complementary adapters on the chip is amplified to form a double strand. The unbound complementary single strands are washed away, leaving the amplified strands. Their free ends can hybridize with other nearby adapter primers, forming a bridge structure. After complementary amplification, they are denatured into single strands, which then interact with other nearby adapter primers to form a bridge, serving as a template for the next round of amplification. After repeated amplification, each single-copy DNA molecule is amplified nearly a thousandfold, resulting in a monoclonal DNA cluster.

[0006] DNA nanoball amplification primarily utilizes MGI's various DNBSEQ sequencing platforms. DNA nanoball amplification is based on rolling circle amplification, enabling template amplification in solution. To achieve rolling circle amplification, the DNBSEQ library is prepared as a single-stranded circle. This single-stranded circle library serves as a template for rolling circle replication in a reaction system with a strand-displacing polymerase, ultimately yielding hundreds of copies of a linear DNA replication strand. These strands entangle to form a nanoball, significantly enhancing signal intensity. DNB nanoball amplification is a linear, not exponential, amplification process.

[0007] The above three major existing technologies have indeed solved the technical difficulties of template amplification in high-throughput sequencing, but they also have the following shortcomings:

[0008] Bead-emulsion PCR amplification: DNA sequencing templates can be amplified to thousands of copies, but to maintain this high copy number, the beads are large, typically measuring micrometers in diameter, ranging from 1 to tens of micrometers, which impacts sequencing throughput. Using small beads increases the likelihood of a single droplet containing two or more beads, impacting sequencing accuracy. Furthermore, droplet preparation requires specialized instrumentation, which is costly and complex.

[0009] Solid-phase bridge PCR amplification: The resulting DNA sequencing template (or cluster) has a high copy number, nearly a thousand copies. However, increasing the copy number requires a larger chip area, which hinders improvements in sequencing throughput and sequencing length. Furthermore, the sequencing chip must be pre-grown with adapters, placing high demands on the chip.

[0010] DNA nanoball amplification: Linear amplification results in a low copy number of the DNA template within the DNA nanoball, typically a few hundred copies. DNA nanoballs have a loose structure, and increasing the copy number significantly reduces their volume, impacting sequencing throughput and sequencing length. Furthermore, increasing the copy number or using long DNA templates requires high processivity from the strand-displacing polymerase. The loose structure of DNA nanoballs makes them susceptible to disruption before loading onto the chip, requiring extreme caution and increasing operational complexity.

[0011] Therefore, the existing technology still needs to be improved in terms of the problem of template amplification during sequencing.

[0012] Summary of the Invention

[0013] The present disclosure aims to solve one of the technical problems in the related art at least to a certain extent.

[0014] One aspect of the present disclosure provides a nucleic acid amplification method. According to an embodiment of the present disclosure, the nucleic acid amplification method includes:

[0015] (A) connecting the nucleic acid to a known sequence to obtain the nucleic acid to be amplified;

[0016] (B) designing a primer pair based on a known sequence contained in the nucleic acid to be amplified;

[0017] (C) performing an amplification and ligation reaction using the nucleic acid to be amplified and the primer pair to obtain a first double-stranded DNA multi-copy concatemer.

[0018] One aspect of the present disclosure provides a nucleic acid amplification method. According to an embodiment of the present disclosure, the nucleic acid amplification method includes:

[0019] (a) amplifying a target nucleic acid using an amplification primer to obtain an amplification product, wherein the 5' end of the amplification primer has a known sequence;

[0020] (d) designing a primer pair based on the known sequence contained in the amplified product, wherein at least one primer in the primer pair contains a restriction enzyme cleavage site;

[0021] (c) performing an amplification and ligation reaction using the amplified product and the primers to obtain a first double-stranded DNA multi-copy concatemer.

[0022] The nucleic acid amplification method disclosed in this invention is easy to operate and does not require complex instrumentation. The resulting concatenated single-stranded DNA copies are compact, have a large copy number, and are structurally stable. This effectively addresses the issues of DNA nanospheres obtained using rolling circle amplification, which are difficult to operate, have a loose structure, are bulky, and have low amplification efficiency. If applied to sequencing, this method can conserve chip space and reduce costs, while also further improving sequencing accuracy due to its large copy number.

[0023] According to the embodiment of the present disclosure, when any one primer in the primer pair contains enzyme cleavage site 1, the other primer does not contain an enzyme cleavage site, or contains an enzyme cleavage site 2 different from the enzyme cleavage site 1;

[0024] in,

[0025] The enzyme cleavage site 1 is cleaved by enzyme 1, and the enzyme cleavage site 2 is cleaved by enzyme 2, and enzyme 1 and enzyme 2 are different.

[0026] According to an embodiment of the present disclosure, step (c) or (c) further comprises,

[0027] (D) When any one primer in the primer pair contains restriction enzyme cleavage site 1 and the other primer does not contain restriction enzyme cleavage site 1, enzyme 1 is used to cut the restriction enzyme cleavage site 1 contained in the double-stranded DNA multi-copy concatemer to form a single-stranded gap in the double-stranded DNA loop, and the strand with the gap is digested with a digestive enzyme to obtain a first single-stranded DNA multi-copy concatemer.

[0028] According to an embodiment of the present disclosure, the 5' ends of the primers in the primer pair all contain phosphorylation modifications.

[0029] The 5' end of the primer in the primer pair is phosphorylated to facilitate the ligase to connect the 3' end of the chain amplification product to the 5' end of the primer to form a loop.

[0030] According to the embodiments of the present disclosure, the two primers in the primer pair have at least partially overlapping complementary segments, and the Tm value of the overlapping complementary segments is lower than the Tm value of the two primers. This ensures that during annealing, the primers preferentially bind to the template rather than forming dimers between the two primers.

[0031] According to an embodiment of the present disclosure, there is no overlapping complementary fragment between the two primers in the primer pair.

[0032] According to an embodiment of the present disclosure, the enzyme cleavage site includes a modified ribonucleotide or deoxyribonucleotide that can be cleaved by the enzyme.

[0033] According to an embodiment of the present disclosure, at least one primer in the primer pair carries a modified ribonucleotide or deoxyribonucleotide, and the restriction site is cut by the enzyme to form a gap on any one circular DNA in the double-stranded DNA concatemer.

[0034] According to an embodiment of the present disclosure, the enzyme cleavage site includes at least one selected from ribonucleotides, uracil deoxyribonucleotides, 5,6-dihydroxythymine deoxyribonucleotides, 5-hydroxyuracil (5-hydroxyuracil) deoxyribonucleotides, 5-hydroxymethyluracil (5-hydroxymethyluracil) deoxyribonucleotides, and 5-formyluracil (5-formyluracil) deoxyribonucleotides.

[0035] According to an embodiment of the present disclosure, the nucleic acid includes at least one selected from linear DNA and circular DNA.

[0036] According to an embodiment of the present disclosure, the nucleic acid includes a chain DNA library and / or a circular DNA library.

[0037] According to an embodiment of the present disclosure, the circular DNA library is a single-stranded circular DNA library.

[0038] According to an embodiment of the present disclosure, the circular DNA library is a double-stranded circular DNA library.

[0039] According to an embodiment of the present disclosure, when the nucleic acid is a chain DNA, step (A) further comprises performing a cyclization reaction on the nucleic acid connected to the known sequence to obtain a cyclized nucleic acid to be amplified.

[0040] In step (A) of the present invention, the nucleic acid to be amplified obtained after connecting the nucleic acid to the known sequence is preferably a circularized nucleic acid, which facilitates the connection of the 5' end and the 3' end of the amplified chain into a ring and improves the connection efficiency.

[0041] According to an embodiment of the present disclosure, the two primers in the primer pair contain enzyme cleavage site 1 and enzyme cleavage site 2, respectively, and the enzyme cleavage site 2 is cleaved by enzyme 2.

[0042] According to an embodiment of the present disclosure, the nucleic acid amplification method further comprises, after step (D):

[0043] (E) using the first single-stranded DNA multi-copy concatemer as a template, performing an amplification and ligation reaction using the primer in the primer pair to obtain a second double-stranded DNA multi-copy concatemer;

[0044] (F) using enzyme 2 to cut the enzyme cleavage site 2 contained in the second double-stranded DNA multi-copy concatemer to form a gapped single strand in the second double-stranded DNA multi-copy concatemer, and digesting the gapped single strand with a digestive enzyme to obtain a second single-stranded DNA multi-copy concatemer.

[0045] According to an embodiment of the present disclosure, the nucleic acid amplification method further comprises, after step (D):

[0046] (G) using the primer in the primer pair that binds to the first multi-copy concatemer of single-stranded DNA and an enzyme with strand displacement ability, to perform an amplification, displacement and ligation reaction on the first multi-copy concatemer of single-stranded DNA in step (D) to obtain a second multi-copy concatemer of single-stranded DNA.

[0047] According to an embodiment of the present disclosure, the amplification and ligation reaction system contains a DNA polymerase that does not have 5'-3' exonuclease activity. The DNA polymerase does not have 5' to 3' exonuclease activity, which can ensure the efficiency of amplification and prevent the amplified product from being cleaved by enzymes.

[0048] According to an embodiment of the present disclosure, the amplification and ligation reaction system contains a DNA polymerase, and the DNA polymerase includes at least one selected from KAPA HiFi HotStart DNA Polymerase, KAPA HiFi HotStart Uracil+Polymerase, BGI Golden High-Fidelity Polymerase, and BGI Platinum HiFi Hotstart Polymerase.

[0049] According to an embodiment of the present disclosure, the amplification and ligation reaction system contains a high-temperature resistant DNA ligase.

[0050] According to an embodiment of the present disclosure, the thermostable DNA ligase comprises at least one selected from Taq ligase and AMP ligase.

[0051] According to an embodiment of the present disclosure, the amplification and ligation reaction system contains a high-temperature-insensitive DNA ligase.

[0052] According to an embodiment of the present disclosure, the thermostable DNA ligase includes at least one selected from T4 DNA ligase, E. coli DNA Ligase, T3 DNA Ligase, and T7 DNA Ligase.

[0053] According to an embodiment of the present disclosure, when the amplification and ligation reaction system contains a thermostable DNA ligase, it further includes a DNA helicase and a single-strand binding protein. The single-strand binding protein contained in the system can prevent the unwound DNA from self-pairing.

[0054] According to an embodiment of the present disclosure, the enzyme 1 and the enzyme 2 include enzymes capable of cleaving the ribonucleotides and / or uracil deoxyribonucleotides.

[0055] According to an embodiment of the present disclosure, the enzyme 1 and enzyme 2 include at least one selected from USER enzyme, RNase H, UDG, RNase HII, hSMUG1, APE1, Endonuclease VIII, Endonuclease III (Nth), and enzyme 1 and enzyme 2 are different enzymes.

[0056] According to an embodiment of the present disclosure, the digestive enzyme comprises an enzyme capable of digesting linear DNA.

[0057] According to an embodiment of the present disclosure, the digestive enzyme comprises a DNA exonuclease.

[0058] According to an embodiment of the present disclosure, the DNA exonuclease includes at least one selected from DNA exonuclease I, DNA exonuclease III, T5 Exonuclease, Exonuclease T, Exonuclease VII, and Lambda Exonuclease.

[0059] Another aspect of the present disclosure provides a method for increasing the copy number of a sequencing template. According to an embodiment of the present disclosure, the method comprises:

[0060] Using a single-stranded circular DNA library and / or a double-stranded circular DNA library with sequencing adapter sequences as templates, the aforementioned nucleic acid amplification method is used to obtain multiple single-stranded DNA multi-copy concatemers to achieve multi-copy amplification of the sequencing template.

[0061] The above-mentioned nucleic acid amplification method is used to perform multi-copy amplification of the sequencing template. The resulting single-stranded DNA multi-copy concatemer is used as the base signal acquisition unit in sequencing and loaded onto the sequencing chip. This method has the following advantages: the DNA multi-copy concatemer has a large number of DNA sequencing template copies and is compact, thus occupying a small area of ​​the sequencing chip or flow cell. Increasing the number of DNA sequencing template copies does not affect its volume, and the fragment length of the DNA sequencing template has little effect on the volume of the DNA multi-copy concatemer, which is conducive to improving sequencing read length. Furthermore, the DNA multi-copy concatemer has a stable structure, which is convenient for operation and long-term storage, and has low requirements for the DNA polymerase used in the reaction.

[0062] Another aspect of the present disclosure provides a single-stranded DNA multi-copy concatemer. According to an embodiment of the present disclosure, the single-stranded DNA multi-copy concatemer is prepared by the aforementioned nucleic acid amplification method.

[0063] According to an embodiment of the present disclosure, the multi-copy concatemer of single-stranded DNA contains at least two single-stranded DNA circular molecules, wherein the single-stranded DNA circular molecules include two known nucleic acid sequences and a nucleic acid fragment to be detected.

[0064] According to an embodiment of the present disclosure, the single-stranded DNA circular molecule further includes a tag sequence, and the tag sequence is located between the two known nucleic acid sequences.

[0065] According to the embodiment of the present disclosure, the multi-copy concatemer of single-stranded DNA further comprises multiple linear DNA strands, the 5' end of each linear DNA strand is an amplification primer capable of hybridizing to a known sequence and a nucleic acid sequence complementary to the nucleic acid fragment to be detected.

[0066] According to an embodiment of the present disclosure, the 5' end of the linear DNA single strand is free, and the 3' end is hybridized to the concatemer.

[0067] Another aspect of the present disclosure provides a nucleic acid composite molecule, which includes the above-mentioned single-stranded DNA multi-copy concatemer and multiple linear DNA single chains, wherein the 5' end of the linear DNA single chain is an amplification primer that can hybridize to a known sequence and a nucleic acid sequence complementary to the nucleic acid fragment to be detected.

[0068] According to an embodiment of the present disclosure, the 5' end of the linear DNA single strand is free, and the 3' end is hybridized to the concatemer.

[0069] Another aspect of the present disclosure provides uses of the single-stranded DNA multi-copy concatemer prepared by the aforementioned nucleic acid amplification method, the aforementioned single-stranded DNA multi-copy concatemer, and the aforementioned nucleic acid composite molecule in sequencing.

[0070] Another aspect of the present disclosure provides a method for producing a second strand, comprising using the aforementioned single-stranded DNA multi-copy concatemer as a template, hybridizing with a primer that is fully or partially complementary to a known nucleic acid sequence, and performing a rolling circle amplification reaction under the action of a polymerase to generate a second strand.

[0071] According to an embodiment of the present disclosure, the primer is immobilized on a solid support, and the solid support may include a material selected from the group consisting of: magnetic beads, polymers, hydrogels, metals, or chips.

[0072] According to an embodiment of the present disclosure, the primer is free in solution.

[0073] The aforementioned multi-copy concatemers of single-stranded DNA can be used as base signal acquisition units and serially loaded onto sequencing chips. These concatemers are compact, feature a large number of copies, and possess a stable structure, effectively resolving the issues inherent in rolling circle amplification (RCA) DNA nanospheres, which are difficult to manipulate, have a loose structure, are bulky, and exhibit low amplification efficiency. Application of these concatemers in sequencing can conserve chip space, reduce costs, and improve accuracy.

[0074] Another aspect of the present disclosure provides a single-stranded DNA multi-copy concatemer prepared by the aforementioned nucleic acid amplification method, and uses of the aforementioned single-stranded DNA multi-copy concatemer in template amplification and micro-amplification.

[0075] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0077] FIG1 shows a schematic flow chart of a nucleic acid amplification method according to one embodiment of the present disclosure;

[0078] FIG2 shows a schematic flow chart of a nucleic acid amplification method according to another embodiment of the present disclosure;

[0079] FIG3 shows a technical roadmap of a nucleic acid amplification method according to an embodiment of the present disclosure;

[0080] FIG4 shows a diagram of a method for forming multiple copies of single-stranded DNA concatemers according to an embodiment of the present disclosure;

[0081] FIG5 shows a method for forming multiple copies of single-stranded DNA concatemers from a chained nucleic acid template according to one embodiment of the present disclosure;

[0082] FIG6 shows an electrophoresis diagram of a DNA multi-copy concatemer according to an embodiment of the present disclosure;

[0083] FIG7 shows an atomic force micrograph of a chip after DNA multi-copy concatemer sequencing according to an embodiment of the present disclosure;

[0084] FIG8 is a schematic diagram showing a comparison of the theoretical sizes of DNA multi-copy concatemers and DNA nanoballs obtained from templates of the same length according to an embodiment of the present disclosure.

[0085] Detailed Description of the Invention

[0086] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0087] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0088] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0089] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0090] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0091] Definitions and Explanations of Terms

[0092] In the present disclosure, the term "template" refers to the nucleic acid molecule to be detected, which means a polymer of nucleotides of a certain length, which may include one or more components of ribonucleotides, deoxyribonucleotides, analogs or derivatives of ribonucleotides or deoxyribonucleotides; including single-stranded or double-stranded nucleic acid molecules.

[0093] In the present disclosure, the term "sequencing" may also be referred to as "nucleic acid sequencing" or "gene sequencing", which refers to the determination of the order of base arrangement in a nucleic acid sequence; it includes double-end sequencing, single-end sequencing and / or paired-end sequencing, etc. The so-called double-end sequencing or paired-end sequencing can refer to the reading of any two segments or two parts that do not completely overlap of the same nucleic acid molecule; the so-called sequencing includes the process of binding nucleotides (including nucleotide analogs) to templates and collecting corresponding reaction signals.

[0094] In the present disclosure, "nucleotide" refers to the four natural nucleotides (dATP, dCTP, dGTP, dTTP) or their derivatives, unless otherwise specifically defined.

[0095] In the present disclosure, the term "primer" refers to an oligonucleotide or nucleic acid molecule that can hybridize to a target sequence of interest; a primer is a single-stranded oligonucleotide or polynucleotide.

[0096] In the present disclosure, the term "base", also known as nucleobase, nitrogenous base, includes natural bases, non-natural bases and base analogs. Among them, natural bases include adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U); non-natural bases include locked nucleic acids (LNA) and bridged nucleic acids (BNA); base analogs include hypoxanthine, deazaadenine, deazaguanine, deazahypoxanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine. In the present disclosure, since the nucleotide type is determined by the base type, the base type can be used to represent the nucleotide type in the present disclosure.

[0097] In this disclosure, the term "rolling circle amplification" refers to a recently developed isothermal nucleic acid amplification method. Using circular DNA as a template, a short DNA primer (complementary to a portion of the circular template) is enzymatically converted into single-stranded DNA containing hundreds or thousands of repeats of the template-complementary fragment.

[0098] In the present disclosure, the term "DNA multi-copy concatemer" refers to the use of a single-stranded DNA circular library or a double-stranded circular DNA library to perform amplification and ligation reaction to form hundreds or thousands of copies of internally linked covalently chained circular DNA.

[0099] As used herein, the term "DNA helicase" refers to a class of enzymes that unwind hydrogen bonds, using ATP hydrolysis to provide energy for DNA unwinding. They often rely on the presence of single strands and can recognize the single-stranded structure of replication forks. They generally catalyze the unwinding of double-stranded DNA or RNA during DNA or RNA replication.

[0100] In the present disclosure, the term "single-strand binding protein", also known as DNA binding protein (SSB, single strand DNA-binding protein), is necessary for DNA replication. Single-strand binding protein is not an enzyme. The single-strand binding protein in Escherichia coli cells is composed of 4 identical subunits with a relative molecular mass of 74,000. The span of binding to single-stranded DNA is about 32 nucleotide units. After the DNA is unwound, the DNA molecules tend to bind into double strands as long as the bases are paired. SSB binds to the single-stranded region produced by the helicase moving forward in the direction of the replication fork, preventing the newly formed single-stranded DNA from re-pairing to form double-stranded DNA or proteins degraded by nucleases. SSB exhibits a synergistic effect when acting, ensuring that SSB continues to bind to the downstream segment. Unlike polymerase, it does not move forward in the direction of replication, but constantly binds and detaches.

[0101] As used herein, the term "exonuclease" refers to a class of enzymes that sequentially catalyze the hydrolysis of 3,5-phosphodiester bonds from one end of a polynucleotide chain, degrading nucleotides. The final product of this hydrolysis is a single nucleotide (dNMP for DNA and NMP for RNA). Based on the nature of their action, they can be divided into single-stranded exonucleases and double-stranded exonucleases.

[0102] In this disclosure, the term "Tm" refers to the temperature at which the absorbance reaches half its maximum value, also known as the melting point or melting point of DNA. The Tm value is the melting temperature of DNA and is the temperature at which UV absorbance reaches half its maximum value during DNA denaturation. DNA sequences have different Tm values; higher CG content in the DNA indicates a higher Tm value.

[0103] In the present disclosure, the term "base signal acquisition unit" refers to a monoclonal population formed after a single molecule template is amplified hundreds or thousands of times, which can amplify the fluorescent signal of the sequenced base. The collected fluorescent signal represents the signal of the original single molecule template.

[0104] As used herein, the term "strand displacement" refers to the chemically modified restriction enzyme cleavage site in a nucleic acid, which, after cleavage, nicks the DNA strand. DNA polymerase then extends the 3' end of the nick and displaces the next DNA strand. The displaced single-stranded DNA can bind to a primer and be extended into a double-stranded DNA strand by the DNA polymerase. This process is repeated repeatedly, resulting in efficient amplification of the nucleic acid sequence.

[0105] In the present disclosure, the term "linker" refers to a known short base sequence.

[0106] Nucleic acid amplification methods

[0107] According to some specific embodiments of the present disclosure, the present disclosure provides a method for nucleic acid amplification, as shown in FIG1 , comprising:

[0108] S110, connecting the nucleic acid to a known sequence to obtain the nucleic acid to be amplified;

[0109] S120, designing a primer pair based on the known sequence contained in the nucleic acid to be amplified;

[0110] S130, performing an amplification and ligation reaction using the nucleic acid to be amplified and the primer pair to obtain multiple copies of a double-stranded DNA concatemer.

[0111] According to some specific embodiments of the present disclosure, the present disclosure provides a method for nucleic acid amplification, as shown in FIG2 , comprising:

[0112] S140, amplifying the target nucleic acid using an amplification primer to obtain an amplification product, wherein the 5' end of the amplification primer has a known sequence;

[0113] S150, designing a primer pair based on the known sequence contained in the amplified product, wherein at least one primer in the primer pair contains a restriction enzyme cleavage site;

[0114] S160, performing an amplification and ligation reaction using the amplified product and the primers to obtain a first double-stranded DNA multi-copy concatemer.

[0115] According to some more specific embodiments of the present disclosure, the present disclosure provides a method for nucleic acid amplification, as shown in FIG3 , comprising:

[0116] 1) Extract total DNA from samples;

[0117] 2) Using mechanical, ultrasonic, or enzymatic methods, the DNA is broken into fragments of varying sizes, which are the DNA to be tested;

[0118] 3) Adding an adapter sequence to at least one end of the fragment and performing a circularization reaction to obtain a circular DNA library;

[0119] 4) Designing a primer pair based on the linker, phosphorylating the 5' ends of both primers, and ensuring that at least one primer in the primer pair has a base modification;

[0120] 5) placing the obtained primers and the obtained circularized DNA into a system for amplification and ligation reaction, ultimately obtaining multiple copies of double-stranded DNA concatemers;

[0121] 6) The obtained multi-copy concatemer DNA is cut with a digestive enzyme that can recognize the modified site in primer 2, so that one of the circular DNAs becomes a chain, and then digested with a single-stranded DNA digestive enzyme to finally obtain multi-copy concatemer single-stranded DNA.

[0122] According to some embodiments of the present disclosure, the 5' ends of the primers in the primer pair are all phosphorylated. "The 5' ends of the primers in the primer pair are all phosphorylated" means that the 5' end of each primer in the primer pair is phosphorylated, which facilitates ligation of the 3' end of the chain amplification product to the 5' end of the primer to form a loop.

[0123] According to some embodiments of the present disclosure, there may be no overlapping complementary segments between the two primers in the primer pair, or the two primers in the primer pair may have at least partially overlapping complementary segments. There is no particular limitation on the length of the overlapping complementary segments, and it can be adjusted as needed. It is also necessary to ensure that the Tm value of the overlapping complementary segment is lower than the Tm value of the two primers. Thus, it can be ensured that during annealing, the primers preferentially bind to the template rather than forming a dimer between the two primers.

[0124] According to some embodiments of the present disclosure, the enzyme cleavage site includes a ribonucleotide or deoxyribonucleotide that can be cut by the enzyme. The enzyme cleavage site includes at least one selected from ribonucleotides, uracil deoxyribonucleotides, 5,6-dihydroxythymine deoxyribonucleotides, 5-hydroxyuracil deoxyribonucleotides, 5-hydroxymethyluracil deoxyribonucleotides, and 5-formyluracil deoxyribonucleotides. It should be noted that the "enzyme cleavage site" described in the present invention is not limited to the above types, and can also be other types of enzyme cleavage sites. As long as the specific cutting of the enzyme can be achieved, this type of enzyme cleavage site is included in the type of enzyme cleavage site of the present invention.

[0125] According to some embodiments of the present disclosure, at least one primer in the primer pair contains a restriction enzyme cleavage site that can be cleaved by an enzyme, thereby forming a gap in any one of the circular DNA strands in the double-stranded DNA concatemer. For example, one of the primers in the primer pair contains a restriction enzyme cleavage site that can be cleaved by an enzyme, thereby forming a gapped strand on the strand of the double-stranded DNA concatemer containing the restriction enzyme cleavage site; or, the two primers in the primer pair contain different restriction enzyme cleavage sites, and corresponding enzymes are used to cleave the restriction enzyme cleavage sites of the double-stranded DNA concatemer as needed to form a gapped strand.

[0126] According to some embodiments of the present disclosure, when the nucleic acid is a chain DNA, step (A) further comprises cyclizing the nucleic acid linked to the known sequence to obtain a circularized nucleic acid to be amplified. In step (A) of the present invention, the nucleic acid to be amplified obtained after the nucleic acid is linked to the known sequence is preferably a circularized nucleic acid, which facilitates the ligation of the 5' and 3' ends of the amplified chain into a ring, thereby improving ligation efficiency.

[0127] According to some embodiments of the present disclosure, when the nucleic acid is circular DNA, it is necessary to first linearize the circular DNA to obtain chain DNA, and then add known linker sequences to its two ends for further circularization reaction to obtain the circularized nucleic acid to be amplified.

[0128] According to some embodiments of the present disclosure, another reverse single-stranded DNA multi-copy concatemer following the first single-stranded DNA multi-copy concatemer in step (c) or (D) can be obtained by the following method:

[0129] Method 1

[0130] The two primers used to prepare multi-copy DNA concatemers have different types of restriction sites. For example, primer 1 contains uracil deoxyribonucleotides, while primer 2 contains ribonucleotides. When preparing a single-stranded DNA multi-copy concatemer, after the amplification reaction is complete, RNase H is used to remove the ribonucleotides in primer 2, creating a nick in the strand amplified by primer 2. This nick is then removed using a DNA digestion enzyme, yielding the first single-stranded DNA multi-copy concatemer. After sequencing the first strand, the complementary sequence generated during the sequencing process is eluted. Amplification is then performed using primer 2, followed by a ligation reaction after one cycle, yielding a second-stranded DNA multi-copy concatemer. The first strand is then nicked using USER enzyme, and the entire single-stranded DNA multi-copy concatemer is then removed using a DNA digestion enzyme. Finally, a single-stranded, two-stranded DNA multi-copy concatemer is obtained.

[0131] Method 2

[0132] After step (C), enzyme 2 is used to cut the enzyme cleavage site 2 contained in the double-stranded DNA multi-copy concatemer to form a gapped single strand in the double-stranded DNA loop, and the gapped single strand is digested with a digestive enzyme to obtain a second single-stranded DNA multi-copy concatemer.

[0133] Method 3

[0134] The multi-copy concatemer DNA obtained in step (c) or (D) is used as a template, and primers complementary to the universal linker sequence of the first single-stranded DNA multi-copy concatemer are used for amplification using the Phi29 enzyme. Relying on the chain displacement ability of the Phi29 enzyme, after one circle of amplification on the single-stranded loop, a second single-stranded DNA multi-copy concatemer complementary to the multi-copy concatemer is displaced.

[0135] According to some embodiments of the present disclosure, the enzyme 1 and enzyme 2 respectively include at least one selected from USER enzyme, RNase H, UDG, RNase HII, and hSMUG1, and enzyme 1 and enzyme 2 are different enzymes.

[0136] According to an embodiment of the present disclosure, the amplification and ligation reaction system contains a DNA polymerase that does not have 5'-3' exonuclease activity. The DNA polymerase does not have 5' to 3' exonuclease activity, which can ensure the efficiency of amplification and prevent the amplified product from being cleaved by enzymes.

[0137] According to an embodiment of the present disclosure, the amplification and ligation reaction system contains a DNA polymerase, and the DNA polymerase includes at least one selected from KAPA HiFi HotStart DNA Polymerase, KAPA HiFi HotStart Uracil+Polymerase, BGI Golden High-Fidelity Polymerase, and BGI Platinum HiFi Hotstart Polymerase.

[0138] According to an embodiment of the present disclosure, the amplification and ligation reaction system contains a high-temperature resistant DNA ligase.

[0139] According to an embodiment of the present disclosure, the thermostable DNA ligase comprises at least one selected from Taq ligase and AMP ligase.

[0140] According to an embodiment of the present disclosure, the amplification and ligation reaction system contains a high-temperature-insensitive DNA ligase.

[0141] According to an embodiment of the present disclosure, the thermostable DNA ligase is selected from at least one of T4 DNA ligase, E. coli DNA Ligase, T3 DNA Ligase, and T7 DNA Ligase.

[0142] According to an embodiment of the present disclosure, when the amplification and ligation reaction system contains a thermostable DNA ligase, it further includes a DNA helicase and a single-strand binding protein. The single-strand binding protein contained in the system can prevent the unwound DNA from self-pairing.

[0143] According to an embodiment of the present disclosure, the enzyme 1 and the enzyme 2 include enzymes capable of cleaving the ribonucleotides and / or uracil deoxyribonucleotides.

[0144] According to an embodiment of the present disclosure, the enzyme 1 and enzyme 2 include at least one selected from USER enzyme, RNase H, UDG, RNase HII, hSMUG1, APE1, Endonuclease VIII, Endonuclease III (Nth), and enzyme 1 and enzyme 2 are different enzymes.

[0145] According to an embodiment of the present disclosure, the digestive enzyme comprises an enzyme capable of digesting single-stranded DNA.

[0146] According to an embodiment of the present disclosure, the digestive enzyme comprises a DNA exonuclease.

[0147] According to an embodiment of the present disclosure, the DNA exonuclease includes at least one selected from DNA exonuclease I, DNA exonuclease III, T5 Exonuclease, Exonuclease T, Exonuclease VII, and Lambda Exonuclease.

[0148] According to some more specific embodiments of the present disclosure, the method for nucleic acid amplification ( FIG. 4 ) comprises:

[0149] 1) A single-stranded circular DNA library or a double-stranded circular DNA library with known linker sequences is used as a template. Amplification primers are designed based on the known linker. The two primers may or may not have overlapping complementary regions (the Tm value of the complementary fragment must be lower than the Tm value of both primers). The 5' ends of the primers are phosphorylated, and one of the primers contains a modified nucleotide that can be subsequently removed by the corresponding enzyme, such as a uracil deoxyribonucleotide or a ribonucleotide.

[0150] 2) Amplification and ligation reaction are performed on the single-stranded circular DNA library or double-stranded circular DNA library template, and the amplification and ligation reaction are performed in a reaction system. The reaction system includes amplification primers, DNA polymerase, DNA ligase, dNTP, Mg 2+ ions, etc., where the DNA polymerase lacks 5'-3' exonuclease activity, such as KAPA HiFi HotStart DNA Polymerase or KAPA HiFi HotStart Uracil+Polymerase. The DNA ligase can be a thermostable ligase, such as Taq ligase or AMPligase. Alternatively, a thermostable ligase, such as T4 DNA ligase, can be used. When using a thermostable ligase, an isothermal amplification system is employed, which may include a DNA helicase and a single-stranded binding protein.

[0151] 3) The template reacts in an amplification and ligation reaction system, undergoing denaturation, annealing, extension, and ligation. Annealing, extension, and ligation can be performed at a uniform temperature. If isothermal amplification is used, denaturation, annealing, extension, and ligation can all be performed at the same temperature, and DNA helicase can be used to achieve the effect of DNA denaturation and unwinding. After the primer anneals to the template, DNA polymerase extends the primer, stopping when it reaches the 5' end of the primer. DNA ligase then ligates the 3' end of the extended chain to the 5' end of the primer itself, forming a covalent double-stranded circular DNA. The two strands of the covalent double-stranded circular DNA are interlinked, and even denaturation cannot separate the two strands, remaining entangled. When the amplification and ligation cycles are repeated again, all products remain interlinked and cannot separate, ultimately resulting in multiple copies of the double-stranded DNA from the same template.

[0152] 4) The resulting double-stranded multi-copy DNA concatemer product is digested to obtain single-stranded multi-copy DNA concatemers. Specifically, a series of digestive enzymes are used to remove modifications such as uracil deoxyribonucleotides or ribonucleotides on one of the primers, such as USER enzyme to remove U bases and RNase H to remove ribonucleotides. A nick is created in one of the double-stranded DNA strands, and then single-stranded DNA digestive enzymes, such as DNA exonuclease I or DNA exonuclease III, are used to digest the nicked single strand. These digestion reactions can occur in the same reaction system or in separate reactions. Purification of the digested product yields single-stranded multi-copy DNA concatemers.

[0153] According to some specific embodiments of the present disclosure, the present disclosure proposes a method for producing a second chain, which includes using multiple copies of a single-stranded DNA concatemer as a template, hybridizing with a primer that is fully or partially complementary to a known nucleic acid sequence, and performing a rolling circle amplification reaction under the action of a polymerase to generate a second chain.

[0154] According to an embodiment of the present disclosure, the primer is immobilized on a solid support, and the solid support may be selected from the group consisting of: magnetic beads, polymers, hydrogels, metals, or chips.

[0155] According to an embodiment of the present disclosure, the primer is free in solution.

[0156] Applications in sequencing

[0157] According to the embodiment of the present disclosure, a random base sequence (label sequence) is added between the known adapters at both ends of the template to be amplified, and the above-mentioned nucleic acid amplification method is used to obtain a multi-copy concatemer of single-stranded DNA with two known adapter sequences, a label sequence, and a sequence to be tested as a sequencing template (Figure 5). This collection of hundreds or thousands of copies is loaded onto a sequencing chip as a base signal unit.

[0158] According to the embodiment of the present disclosure, a primer 1 complementary to the adapter sequence is synthesized, and the 5' end of the primer 1 is paired with the adapter sequence using the principle of base complementarity, and a sequencing-by-synthesis reaction is subsequently performed.

[0159] According to a specific embodiment of the present disclosure, after completing the sequencing of the first strand, the second strand can be obtained by the following method:

[0160] Method 1

[0161] Using the current single-strand DNA multi-copy concatemer as a template, the adapter as the primer binding site, and the Phi29 enzyme for amplification, relying on the chain displacement ability of the Phi29 enzyme, after amplifying one circle on the single-stranded loop, a second chain complementary to the multi-copy concatemer is displaced.

[0162] Method 2

[0163] The two primers used to prepare multi-copy DNA concatemers have different types of restriction sites. For example, primer 1 contains uracil deoxyribonucleotides, while primer 2 contains ribonucleotides. To prepare a single-stranded DNA concatemer, after the amplification reaction is complete, RNase H is used to remove the ribonucleotides in primer 2, creating a nick in the strand amplified by primer 2. This nick is then removed using a DNA digestion enzyme, yielding a single-stranded, multi-copy DNA concatemer. After sequencing the first strand, the complementary sequence generated during the sequencing process is eluted. Amplification is then performed using primer 2, followed by a ligation reaction after one cycle, yielding a second-stranded, multi-copy DNA concatemer. The first strand is then nicked using USER enzyme, and the entire single-stranded, multi-copy DNA concatemer is removed using a DNA digestion enzyme. Finally, a single-stranded, two-stranded, multi-copy DNA concatemer is obtained.

[0164] The primers 1 and 2 may have different restriction enzyme cleavage sites, regardless of order. In addition to U bases and ribonucleic acid bases, they may also be 5,6-dihydroxythymine, 5-hydroxyuracil, 5-hydroxymethyluracil, and 5-formyluracil. The enzymes 1 and 2 include at least one selected from USER enzyme, RNase H, UDG, RNase HII, and hSMUG1, and the enzymes 1 and 2 are different enzymes.

[0165] The single-stranded DNA multi-copy concatemer obtained by the above-mentioned nucleic acid amplification method is loaded onto a sequencing chip as a base signal acquisition unit in sequencing, which has the following advantages: the DNA sequencing template copy number of the DNA multi-copy concatemer is large and its volume is small, thereby occupying a small area of ​​the sequencing chip or flow cell; the increase in the copy number of the DNA sequencing template will not affect its volume, and the fragment length of the DNA sequencing template has little effect on the volume of the DNA multi-copy concatemer, which is conducive to improving the sequencing read length. In addition, the structure of the DNA multi-copy concatemer is stable, convenient for operation and long-term storage, and has low requirements for the DNA polymerase used in the reaction. The DNA multi-copy concatemer does not need to be reacted on the sequencing chip or flow cell, and has low requirements for the chip. There is no need to generate nanowells on the chip and pre-plant oligonucleotides on the chip.

[0166] The above-mentioned nucleic acid amplification method is applicable not only to sequencing but also to any micro-amplification field. When experimental materials are limited and samples are relatively scarce, the disclosed method can be used to efficiently amplify the sample nucleic acid content without changing the nucleic acid abundance.

[0167] If no specific techniques or conditions are specified in the implementation plan, the techniques or conditions described in the literature in this field or the product instructions shall be followed. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0168] Example 1 Escherichia coli genomic DNA sequencing

[0169] The experimental material in the embodiment is Escherichia coli genomic DNA

[0170] 1. Experimental Procedure

[0171] (1) Prepare the MGIEasy DNA Library Preparation Kit (16RXN) (MGI, Cat. No. 1000006987).

[0172] (2) 100 ng of Escherichia coli genomic DNA was taken and, according to the kit instructions, the DNA was sheared to a main band of 300 to 500 bp in size. After magnetic bead purification, fragment selection, end repair and A addition, adapter ligation, and PCR amplification, the PCR amplification product was finally circularized using the reagents in the single-stranded circularization part of the kit to obtain the final single-stranded circular DNA library suitable for the DNBSEQ sequencing platform;

[0173] (3) Take 5 ng of the prepared single-stranded circular DNA library and transfer it to a 200 μL PCR tube;

[0174] (4) Prepare the amplification and ligation reaction solution on ice according to Table 1;

[0175] Table 1

[0176]

[0177] Primer1:5'P-GCCATGTCGTTCTGTGAGCCAAGG-3'(SEQ ID NO: 1)

[0178] Primer2:5'P-GCTCACAGAA / rC / / rG / / rA / / rC / ATGGCTACGATCCGAC-3'(SEQ ID NO: 2)

[0179] Where r represents ribonucleotide modification and P represents phosphorylation modification.

[0180] (5) Add the prepared reaction solution to the PCR tube containing the single-stranded circular DNA library and add nuclease-free water (ThermoFisher, AM9930) to a final volume of 50 μL. Mix thoroughly using a shaker and centrifuge briefly to remove the liquid from the tube.

[0181] (6) Place the centrifuged PCR tube on a PCR instrument and perform the reaction according to the reaction program in Table 2;

[0182] Table 2

[0183]

[0184] (7) After the reaction is completed, remove the PCR tube, centrifuge it briefly, and place it on an ice box;

[0185] (8) Prepare the amplification and digestion enzyme reaction solution on an ice box according to Table 3;

[0186] Table 3

[0187]

[0188] (9) Add the prepared digestive enzyme reaction solution to the PCR tube from the previous step. Mix thoroughly using a shaker, then centrifuge briefly to remove the liquid to the bottom of the tube.

[0189] (10) Place the centrifuged PCR tube on a PCR instrument and perform the reaction according to the reaction program in Table 4;

[0190] Table 4

[0191] Temperature Time 45℃ Hot Cover On 37℃ 30min 4℃ Hold

[0192] (11) After the reaction is completed, remove the PCR tube from the PCR instrument and centrifuge briefly to collect the reaction solution at the bottom of the tube;

[0193] (12) Transfer the reaction product to a 1.5 mL centrifuge tube. Then add 150 μL of purified magnetic beads, pipette 10 times to mix thoroughly, and incubate at room temperature for 10 min.

[0194] (13) After instant centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 min until the liquid becomes clear. Use a pipette to aspirate and discard the supernatant.

[0195] (14) Add 200 μL of 80% ethanol to a 1.5 mL centrifuge tube and let it stand for 30 seconds to wash the magnetic beads;

[0196] (15) Repeat the previous step;

[0197] (16) Try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the 1.5 mL centrifuge tube briefly, separate it on a magnetic stand, and then use a small-scale pipette to drain the liquid at the bottom of the tube.

[0198] (17) Keep the 1.5 mL centrifuge tube fixed on the magnetic stand, open the 1.5 mL centrifuge tube cap, and dry it at room temperature until the surface of the magnetic beads is free of reflection and cracks;

[0199] (18) Remove the 1.5 mL centrifuge tube from the magnetic stand, add 26 μL of molecular-grade water to elute the DNA, and pipette gently 10 times to mix thoroughly.

[0200] (19) Incubate at room temperature for 5 min;

[0201] (20) Centrifuge briefly, place the 1.5 mL centrifuge tube on a magnetic stand, let it stand for 5 min until the liquid becomes clear, and transfer 25 μL of the supernatant to a new 1.5 mL centrifuge tube;

[0202] (21) Use ssDNA Assay Kit fluorescence quantitative kit, according to the operating instructions of the quantitative kit to quantify the enzyme digestion and purification products;

[0203] (22) Take 2 μL of the product for electrophoresis detection using a 1.5% agarose gel pre-added with SYBR Safe DNA gel stain (Thermo Fisher, S33102) at 110 V for 45 minutes, and then use the Bio-Rad Gel Doc XR+ gel imaging system to take pictures;

[0204] (23) Prepare the MGISEQ-2000 high-throughput sequencing reagent set (SE100) (MGI, 1000012551) and follow the instructions;

[0205] (24) During the loading process, only the DNB loading buffer II in the kit was used. The corresponding volume was pipetted and added to the PCR tube containing the prepared DNA multi-copy concatemer product. The DNA multi-copy concatemer product was then loaded onto the sequencing chip according to the instructions and sequenced.

[0206] (25) After sequencing is completed, atomic force microscopy is used to observe the multi-copy concatemers of DNA loaded on the sequencing chip;

[0207] 2. Experimental Results

[0208] (1) The concentration of concatemer DNA multi-copy obtained after the reaction of the single-stranded DNA sequencing library was consistent with the expected concentration (Table 5);

[0209] Table 5 Single-stranded DNA library input and DNC yield

[0210]

[0211] (2) The electrophoresis diagram (Figure 6) shows that the band of the obtained product is too large (about 160k) and does not run out of the sample well, which is in line with expectations;

[0212] (3) After sequencing using DNBSEQ sequencing reagents and chips, a read length of more than 415M was obtained, and the Q30 quality value was able to reach more than 89%, meeting the expected goal;

[0213] (4) Using an atomic force microscope to observe the chip after sequencing, it can be observed that the modification range is significantly smaller than the 200 nm diameter of the existing sequencing chip (Figure 7, Figure 8).

[0214] In this example, Escherichia coli genomic DNA was used as the material, and the nucleic acid amplification method disclosed in the present invention was adopted. The obtained amplified products were loaded onto a chip for sequencing, and the results were directly quality checked, which was in line with expectations.

[0215] This embodiment is simple to operate throughout the entire process, from obtaining the nucleic acid template to obtaining the amplified product (multiple copies of single-stranded DNA concatemers). The required instruments and consumables are readily available to experimentalists in this field, thereby improving experimental efficiency while reducing experimental costs. Table 1 shows that a large amount of DNC can be obtained with a small amount of DNA library, and Figure 6 shows that the quality of the obtained DNC meets the standards. Therefore, this method can efficiently amplify the nucleic acid template while ensuring the quality of the amplified product. Figures 7 and 8 show that during sequencing, the disclosed method can significantly reduce the chip area occupied. Compared with the existing technology, it can effectively increase the sequencing read length under the chip of the same area. Moreover, because the template amplification is not performed on the chip, the chip requirements are relatively low.

[0216] Example 2 Sequencing of EGFR gene tumor hotspot regions

[0217] The experimental material in the embodiment is human genomic DNA

[0218] 1. Experimental Procedure

[0219] (1) A primer pool for amplifying the tumor hotspot region of the EGFR gene was designed. The primer pool contained 8 pairs of specific oligonucleotide primers (EGFR_1F-EGFR_8F) and (EGFR_1R-EGFR_8R), with an amplicon size of 100 to 200 bp. The sequences of the specific oligonucleotide primers are shown in Table 6 below.

[0220] Table 6

[0221]

[0222] Note: The above-mentioned specific oligonucleotide primers were mixed at a concentration of 2 μM for each specific oligonucleotide primer to obtain a specific oligonucleotide primer pool with a total concentration of 2 μM;

[0223] (2) 5 ng of human genomic DNA was placed in a PCR tube and the PCR reaction system was configured as shown in (Table 7) for PCR reaction. The reaction program was as follows: 94°C for 1 min; 94°C for 30 s, 58°C for 2 min, 72°C for 30 s, 15 cycles; 72°C for 5 min; 4°C∞.

[0224] Table 7

[0225]

[0226] (3) After the reaction, the DNA library was purified using the magnetic beads in the MGIEasy DNA Library Preparation Reagent Kit (16RXN) (MGI, catalog number 1000006987). The purified product was dissolved in 22 μL of elution buffer to obtain the PCR product.

[0227] (4) 300 ng of PCR amplification product was taken and single-stranded circularized using the reagents in the single-stranded circularization section of the MGIEasy DNA Library Preparation Reagent Set (16RXN) (MGI, Catalog No. 1000006987) to obtain a single-stranded circular DNA library suitable for the DNBSEQ sequencing platform;

[0228] (5) Transfer 5 ng of the prepared single-stranded circular DNA library into a 200 μL PCR tube;

[0229] (6) Prepare the amplification and ligation reaction solution on ice according to Table 8;

[0230] Table 8

[0231]

[0232] Primer1:5'P-GCCATGTCGTTCUGUGAGCCAAGG-3'(SEQ ID NO:19)

[0233] Primer2:5'P-GCTCACAGAA / rC / / rG / / rA / / rC / ATGGCTACGATCCGAC-3'(SEQ ID NO:20)

[0234] Where r represents the modification of ribonucleotide; U represents deoxyribonucleotide; and P represents phosphorylation modification.

[0235] (7) Add the prepared reaction solution to the PCR tube containing the single-stranded circular DNA library and add nuclease-free water (ThermoFisher, AM9930) to a final volume of 50 μL. Mix thoroughly using a shaker and centrifuge briefly to remove the liquid from the tube.

[0236] (8) Place the centrifuged PCR tube on a PCR instrument and perform the reaction according to the reaction program in Table 9;

[0237] Table 9

[0238]

[0239] (9) After the reaction is completed, remove the PCR tube, centrifuge it briefly, and place it on an ice box;

[0240] (10) Prepare the amplification digestion enzyme reaction solution on an ice box according to Table 10;

[0241] Table 10

[0242]

[0243] (11) Add the prepared digestive enzyme reaction solution to the PCR tube from the previous step. Mix thoroughly using a shaker, then centrifuge briefly to remove the liquid to the bottom of the tube.

[0244] (12) Place the centrifuged PCR tube on a PCR instrument and perform the reaction according to the reaction program in Table 11;

[0245] Table 11

[0246] Temperature Time 45℃ Hot Cover On 37℃ 30min 4℃ Hold

[0247] (13) After the reaction is completed, remove the PCR tube from the PCR instrument and centrifuge briefly to collect the reaction solution at the bottom of the tube;

[0248] (14) Transfer the reaction product to a 1.5 mL centrifuge tube. Then add 150 μL of purified magnetic beads, pipette 10 times to mix thoroughly, and incubate at room temperature for 10 min.

[0249] (15) After instant centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 min until the liquid becomes clear. Use a pipette to aspirate and discard the supernatant.

[0250] (16) Add 200 μL of 80% ethanol to a 1.5 mL centrifuge tube and let it stand for 30 seconds to wash the magnetic beads;

[0251] (17) Repeat the previous step;

[0252] (18) Try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the 1.5 mL centrifuge tube briefly, separate it on a magnetic stand, and then use a small-scale pipette to drain the liquid at the bottom of the tube.

[0253] (19) Keep the 1.5 mL centrifuge tube fixed on the magnetic stand, open the 1.5 mL centrifuge tube cap, and dry it at room temperature until the surface of the magnetic beads is free of reflection and cracks;

[0254] (20) Remove the 1.5 mL centrifuge tube from the magnetic stand, add 26 μL of molecular-grade water to elute the DNA, and pipette gently 10 times to mix thoroughly.

[0255] (21) Incubate at room temperature for 5 min;

[0256] (22) Centrifuge briefly, place the 1.5 mL centrifuge tube on a magnetic stand, let it stand for 5 min until the liquid becomes clear, and transfer 25 μL of the supernatant to a new 1.5 mL centrifuge tube;

[0257] (23) Use ssDNA Assay Kit fluorescence quantitative kit, according to the operating instructions of the quantitative kit to quantify the enzyme digestion and purification products;

[0258] (24) Prepare the MGISEQ-2000 high-throughput sequencing reagent set (PE100) (MGI, 1000012554) and complete the preparation of wells 1 and 2 of the reagent tank according to the instructions.

[0259] (25) Then, the reagents in wells 5 and 7 of the reagent tank were aspirated and washed twice with molecular-grade water. Then, 5 mL of formamide was added to well 5 and 3 mL of 1 μM primer for synthesizing the second chain was added to well 7.

[0260] Primer for synthesizing the second strand: 5'P-GCTCACAGAACGACATGGCTACGATCCGAC-3' (SEQ ID NO: 21)

[0261] (26) Prepare the digestive enzyme reaction solution according to Table 12 and add it to well 4.

[0262] Table 12

[0263]

[0264] (26) Prepare the enzyme reaction solution for double-strand synthesis according to Table 13 and add it to well 15.

[0265] Table 13

[0266]

[0267] (27) During the loading process, only the DNB loading buffer II in the kit was used. The corresponding volume was pipetted and added to the PCR tube containing the prepared DNA multi-copy concatemer product. The DNA multi-copy concatemer product was loaded onto the sequencing chip and then sequenced.

[0268] (28) After completing the sequencing of the first chain, pump the formamide reagent into well 5, incubate at 85°C for 10 minutes, and then pump the cleaning reagent into well 7 for washing twice; then pump the primer for synthesizing the second chain into well 7, incubate at 65°C for 2 minutes, and then pump the cleaning reagent into well 7 for washing twice; then pump the enzyme reaction solution reagent for synthesizing the second chain into well 15, incubate at 65°C for 10 minutes, and then pump the cleaning reagent into well 15 for washing twice; finally, pump the digestion enzyme reaction solution into well 4, incubate at 37°C for 30 minutes, and finally pump the cleaning reagent into well 4 for washing twice, and finally complete the synthesis of the second chain.

[0269] (29) After completing the second-strand synthesis, the second strand was sequenced according to the subsequent process of the second-strand sequencing kit.

[0270] (30) After sequencing was completed, the obtained sequencing data were statistically analyzed. The data were directly from the sequencer, including the data volume and the Q30 value reflecting the data quality. The data were then subjected to subsequent alignment analysis. The 4M read length sequence data were extracted for analysis and aligned to the human reference genome (hg19) using BWA software. Samtools was used to analyze the data utilization rate, alignment rate, and target region data ratio of the sequencing data.

[0271] 2. Experimental Results

[0272] (1) The concentration of concatemer DNA multi-copy obtained after the reaction of the single-stranded DNA sequencing library was consistent with the expected concentration (Table 14);

[0273] Table 14 Single-stranded DNA library input and DNC yield

[0274]

[0275] (2) After sequencing using DNBSEQ sequencing reagents and chips, a read length of more than 350M was obtained, of which the Q30 quality value of the first chain was more than 89%, and the Q30 quality value of the second chain was more than 80%, achieving the expected goal;

[0276] (4) The data utilization rate, alignment rate, and target region data ratio of the sequencing data were statistically analyzed, and the results were in line with expectations. The results are shown in Table 15.

[0277] Table 15

[0278]

[0279] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0280] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are exemplary and are not to be construed as limitations of the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A method for nucleic acid amplification, It is characterized in that include: (A) connecting the nucleic acid to a known sequence to obtain the nucleic acid to be amplified; (B) designing a primer pair based on a known sequence contained in the nucleic acid to be amplified; (C) performing an amplification and ligation reaction using the nucleic acid to be amplified and the primers to obtain a first double-stranded DNA multi-copy concatemer.

2. A nucleic acid amplification method, It is characterized in that include: (a) amplifying a target nucleic acid using an amplification primer to obtain an amplification product, wherein the 5' end of the amplification primer has a known sequence; (b) designing a primer pair based on a known sequence contained in the amplified product, wherein at least one primer in the primer pair contains a restriction site; (c) performing an amplification and ligation reaction using the amplified product and the primers to obtain a first double-stranded DNA multi-copy concatemer.

3. The method according to claim 1 or 2, It is characterized in that When any one of the primers in the primer pair contains a restriction site 1, the other primer does not contain a restriction site, or contains a restriction site 2 different from the restriction site 1; in, The enzyme cleavage site 1 is cleaved by enzyme 1, and the enzyme cleavage site 2 is cleaved by enzyme 2, and enzyme 1 and enzyme 2 are different.

4. The nucleic acid amplification method according to claim 3, It is characterized in that Step (c) or (c) further comprises, (D) When any one of the primers in the primer pair contains restriction site 1 and the other primer does not contain restriction site, enzyme 1 is used to cut the restriction site 1 contained in the double-stranded DNA multi-copy concatemer to form a single-stranded gap in the double-stranded DNA loop, and the single-stranded gap is digested with a digestive enzyme to obtain a first single-stranded DNA multi-copy concatemer.

5. The method according to any one of claims 1 to 4, It is characterized in that The 5' ends of the primers in the primer pair all contain phosphorylation modification.

6. The method according to any one of claims 1 to 5, It is characterized in that The enzyme cleavage site includes modified ribonucleotides or deoxyribonucleotides that can be cleaved by the enzyme.

7. The method according to any one of claims 1 to 6, It is characterized in that The restriction site includes at least one selected from ribonucleotide, uracil deoxyribonucleotide, 5,6-dihydroxythymine deoxyribonucleotide, 5-hydroxyuracil deoxyribonucleotide, 5-hydroxymethyluracil deoxyribonucleotide, and 5-formyluracil deoxyribonucleotide.

8. The method according to claim 1 or 2, It is characterized in that When the nucleic acid is a chain DNA, step (A) or (a) further comprises performing a cyclization reaction on the nucleic acid connected to the known sequence to obtain a cyclized nucleic acid to be amplified.

9. The method according to any one of claims 4 to 8, It is characterized in that The two primers in the primer pair contain restriction enzyme site 1 and restriction enzyme site 2 respectively, and the restriction enzyme site 2 is cut by enzyme 2. The nucleic acid amplification method further comprises, after step (D): (E) using the first single-stranded DNA multi-copy concatemer as a template, and using the primers in the primer pair to perform an amplification and ligation reaction to obtain a second double-stranded DNA multi-copy concatemer; (F) using enzyme 2 to cut the restriction site 2 contained in the second double-stranded DNA multi-copy concatemer to form a gapped single strand in the second double-stranded DNA multi-copy concatemer, and using a digestive enzyme to digest the gapped single strand to obtain the second single-stranded DNA multi-copy concatemer.

10. The method according to any one of claims 4 to 8, It is characterized in that The nucleic acid amplification method further comprises, after step (D): (G) using the primer in the primer pair that binds to the first single-stranded DNA multi-copy concatemer and an enzyme with strand displacement ability, the first single-stranded DNA multi-copy concatemer in step (D) is subjected to an amplification displacement ligation reaction to obtain a first single-stranded DNA multi-copy concatemer and a second single-stranded complex.

11. A single-stranded DNA multi-copy concatemer, It is characterized in that The single-stranded DNA multi-copy concatemer is prepared by the nucleic acid amplification method according to any one of claims 1 to 10.

12. The single-stranded DNA multi-copy concatemer according to claim 11, It is characterized in that The single-stranded DNA multi-copy concatemer contains at least two single-stranded DNA circular molecules, wherein the single-stranded DNA circular molecules include two known nucleic acid sequences and a nucleic acid fragment to be detected.

13. The single-stranded DNA multi-copy concatemer according to claim 12, It is characterized in that The single-stranded DNA circular molecule further comprises a tag sequence, and the tag sequence is located between the two known nucleic acid sequences.

14. A nucleic acid complex molecule, It is characterized in that The nucleic acid composite molecule comprises a single-stranded DNA multi-copy concatemer and a plurality of linear DNA single strands as described in any one of claims 11-13, wherein the 5' end of the linear DNA single strand is an amplification primer capable of hybridizing to a known sequence and a nucleic acid sequence complementary to the nucleic acid fragment to be detected.

15. The nucleic acid complex molecule according to claim 14, It is characterized in that The 5' end of the linear DNA single strand is free, and the 3' end is hybridized to the concatemer.

16. Use of the single-stranded DNA multi-copy concatemer prepared by the nucleic acid amplification method according to any one of claims 1 to 10, the single-stranded DNA multi-copy concatemer according to any one of claims 11 to 13, and the nucleic acid composite molecule according to claim 14 or 15 in sequencing.

17. A method for generating a second chain, It is characterized in that The single-stranded DNA multi-copy concatemer according to any one of claims 11 to 13 is used as a template, hybridized with a primer that is fully or partially complementary to a known nucleic acid sequence, and a rolling circle amplification reaction is performed under the action of a polymerase to generate a second chain.

18. The method for generating a second strand according to claim 17, It is characterized in that The primers are immobilized on a solid support.

19. The method for generating a second strand according to claim 18, It is characterized in that The solid support may include a material selected from the group consisting of magnetic beads, polymers, hydrogels, metals or chips.

20. The method for generating a second strand according to any one of claims 17 to 19, It is characterized in that The primer is free in solution.

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