WO2024138550A1 - Nucleic acid amplification method and use thereof in sequencing - Google Patents

Abstract

Provided are a nucleic acid amplification method, a single-stranded multi-copy DNA concatemer and the use thereof. The method comprises: linking a nucleic acid with a known sequence to obtain a nucleic acid to be amplified, and performing an amplification ligation reaction using a primer pair designed on the basis of the known sequence contained in the nucleic acid to be amplified, thereby obtaining a first double-stranded multi-copy DNA concatemer, wherein the two primers of the primer pair can comprise different enzyme cleavage sites 1 and 2 respectively; using an enzyme 1 to cleave the enzyme cleavage site 1 contained in the double-stranded multi-copy DNA concatemer so as to form a single-stranded gap in the double-stranded DNA loop, and using a digestive enzyme to digest the single-stranded gap, so as to obtain a first single-stranded multi-copy DNA concatemer; performing an amplification ligation reaction using a primer with the first single-stranded multi-copy DNA concatemer serving as a template so as to obtain a second double-stranded multi-copy DNA concatemer, using an enzyme 2 to cleave the enzyme cleavage site 2 contained in the second double-stranded multi-copy DNA concatemer so as to form a gap single strand in the second double-stranded multi-copy DNA concatemer, and using the digestive enzyme to digest the gap single strand, so as to obtain a second single-stranded multi-copy DNA concatemer; and using the above-mentioned single-stranded multi-copy DNA concatemer for nucleic acid sequencing .

Description

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

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

Background technique

In the field of life science research, sequencing technology has become one of the most commonly used and important research methods. Since Sanger invented the dideoxy chain termination method, the first generation sequencing technology, in 1977, and the promotion of the Human Genome Project, the first generation sequencing technology has greatly improved the speed and throughput of sequencing from the initial isotope labeling plus plate electrophoresis to fluorescent labeling and capillary electrophoresis and automated imaging systems. However, compared with the growing demand for sequencing, the developed first generation sequencing technology still cannot meet the needs. Therefore, the next generation sequencing technology was invented and developed rapidly in a short period of time. The next generation sequencing technology is also called the second generation sequencing technology or massively parallel sequencing technology. Its main feature is that it can sequence a large number of DNA fragments (millions to billions) at the same time. Compared with the first generation of Sanger sequencing, the sequencing speed and throughput have been substantially improved, which can further meet the sequencing needs of scientific research and application. The conventional steps of large-scale parallel sequencing technology are to fragment DNA, add adapters with known sequences to form a so-called library, and then load these DNA fragments with adapters onto the sequencing chip. Each DNA fragment with adapters in the library occupies a certain position on the chip, so that all the sequences on the chip are sequenced at one time, thereby achieving the purpose of large-scale parallel sequencing. In this process, an extremely important step is what we call the sequencing template signal amplification step. This is because, whether relying on fluorescent signals or ion signals, a single copy of the DNA sequence cannot provide sufficient information intensity, so before the DNA sequence is loaded onto the chip, each single copy of the DNA fragment in the library must be amplified. Such amplification is to amplify the single copy of the DNA sequence into a monoclonal multi-copy DNA sequence in a fixed spatial position, generating the so-called base signal acquisition unit to increase the signal intensity and ensure that the signal can be distinguished from the background noise; on the other hand, it helps to form independent reaction centers, so that all reaction centers can react at the same time, collect information, perform signal detection and interpretation, and read the sequence information of the DNA fragments in sequence. The number of copies of the DNA fragment amplified by the template and the size of the space occupied by the multi-copy template have a great impact on the accuracy of sequencing, sequencing read length and sequencing throughput. The more copies there are, the stronger the signal, the higher the signal-to-background noise ratio, and the higher the sequencing accuracy. The more copies there are, the more it can combat the asynchrony between copies of the same type during the sequencing process and improve the read length. The smaller the space occupied by the multi-copy template, the more reaction centers can be arranged on the limited sequencing chip, and the more likely it is to increase the sequencing throughput. Therefore, for large-scale parallel sequencing, template amplification technology is one of the most important core technologies.

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.

Microbead emulsion PCR amplification is mainly used in Roche's 454 sequencing platform and Thermo Fisher's SOLiD and Ion torrent platforms. The specific steps are to mix the DNA library with the adapter sequence with the microbeads with complementary sequences to the adapter, dNTP, primers and DNA polymerase and other reactants, add specific mineral oil and surfactant, and then use an oscillator to oscillate violently to make the reaction system form a stable water-in-oil emulsion. Under ideal conditions, each droplet will contain only one magnetic bead and a single-stranded DNA. By controlling the conditions of this step, at least 10 to the power of 6 ideal droplets can be formed in 1mL of emulsion. After PCR amplification, the surface of each bead is covered with thousands of copies of the same DNA sequence. Subsequently, the emulsion mixture is broken, and the amplified fragments are still bound to the magnetic beads.

Solid-phase bridge PCR amplification is mainly used in various Illumina sequencing platforms. In solid-phase bridge PCR amplification, fragmented DNA is connected to adapters to form a library, and the library with adapters is converted into single strands, which are then diluted to a suitable concentration and added to the sequencing chip. The single-stranded DNA fragments are bound to the sequencing chip through complementary adapters. Because the concentration of the library is low enough after dilution, it can be considered that the library fragments are evenly bound to the chip surface, and the positions where each fragment is bound are far enough apart. The single-stranded DNA hybridized with the complementary adapter on the chip is amplified to form a double strand, and the unfixed complementary single strand is washed away, leaving the amplified chain, whose free end can hybridize with other nearby adapter primers to form a bridge structure. After complementary amplification, it is denatured into a single strand and interacts with other nearby adapter primers again to form a bridge, becoming the template for the next round of amplification and continuing amplification. After repeated amplification, each single copy of the DNA molecule is amplified nearly a thousand times to become a monoclonal DNA cluster.

DNA nanoball amplification mainly uses various DNBSEQ sequencing platforms manufactured by MGI. DNA nanoball amplification is based on rolling circle amplification, which can complete template amplification in solution. In order to achieve rolling circle amplification, the DNBSEQ library will be prepared into a single-stranded circle. Such a single-stranded circle library will be used as a template for rolling circle replication in the reaction system of a polymerase with chain displacement ability, and finally hundreds of copies of linear DNA replication chains will be obtained. This replication chain will be entangled together to form a nanoball, which greatly enhances the signal intensity. DNB nanoball amplification is a linear amplification process, not an exponential amplification.

The above three main existing technologies have indeed solved the technical problems of template amplification in high-throughput sequencing, but they also have the following shortcomings:

Microbead emulsion PCR amplification: The copy number of the DNA sequencing template is high and can be amplified to thousands of copies. However, in order to ensure the copy number, the volume of the microbeads is relatively large, and the diameter is generally in the micrometer range, ranging from 1 micron to tens of microns, which affects the sequencing throughput. The use of small microbeads will increase the number of microbeads contained in a droplet, affecting the accuracy of sequencing. In addition, corresponding instruments are required when preparing droplets, which is costly and complicated to operate.

Solid-phase bridge PCR amplification: The copy number of the obtained DNA sequencing template (or cluster) is relatively high, with nearly a thousand copies. However, if the copy number needs to be increased, a larger chip area will be required, resulting in the inability to increase the sequencing throughput and sequencing length. In addition, the sequencing chip needs to be pre-planted with connectors, which places high demands on the chip.

DNA nanoball amplification: Linear amplification makes the number of DNA template copies in the DNA nanoball not high, generally a few hundred copies. The structure of the DNA nanoball is loose, and increasing the number of copies will greatly affect the volume of the DNA nanoball, affecting the improvement of sequencing throughput and sequencing length. In addition, increasing the number of copies or long-fragment DNA templates requires high continuous synthesis ability of the chain displacement polymerase. The structure of the DNA nanoball is loose, and before it is loaded onto the chip, its structure is easily destroyed, resulting in the need for extra caution in its operation, which increases the complexity of the operation.

Therefore, the existing technology still needs to be improved on the issue of template amplification during sequencing.

Summary of the invention

The present disclosure aims to solve one of the technical problems in the related art at least to some extent.

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 comprises:

(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 primer pair to obtain a first double-stranded DNA multi-copy concatemer.

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 comprises:

(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;

(d) 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.

The nucleic acid amplification method disclosed in the present invention is easy to operate and does not require complex instruments. The obtained single-stranded DNA multiple copies are small in size, large in number of copies, and stable in structure, which effectively solves the problem that the DNA nanoballs obtained by the rolling circle amplification method are difficult to operate, loose in structure, large in size, and low in amplification efficiency. If it is applied to the field of sequencing, it can save the chip usage area, reduce costs, and further improve the accuracy of sequencing due to its large copy number.

According to the embodiments of the present disclosure, when any one primer in the primer pair contains restriction site 1, the other primer does not contain restriction site, or contains restriction site 2 different from 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.

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

(D) When any one of the primers in the primer pair contains a restriction site 1 and the other primer does not contain a 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 strand with the gap is digested with a digestive enzyme to obtain a first single-stranded DNA multi-copy concatemer.

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

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 ring.

According to the embodiments of the present disclosure, the two primers in the primer pair have at least partially overlapping complementary fragments, and the Tm value of the overlapping complementary fragments is lower than the Tm value of the two primers. Thus, it can be ensured that the primers preferentially bind to the template during annealing rather than forming a dimer between the two primers.

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

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.

According to the embodiments 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 of the circular DNAs in the double-stranded DNA concatemer.

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.

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

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

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

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

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

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.

According to an embodiment of the present disclosure, the two primers in the primer pair contain restriction site 1 and restriction site 2, respectively, and the restriction site 2 is cut by enzyme 2.

According to an embodiment of the present disclosure, 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.

According to an embodiment of the present disclosure, 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 second single-stranded DNA multi-copy concatemer.

According to the embodiments of the present disclosure, the amplification and ligation reaction system contains a DNA polymerase, and the DNA polymerase 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 amplification product from being cleaved by enzymes.

According to the embodiments 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.

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

According to an embodiment of the present disclosure, the high temperature resistant DNA ligase includes at least one selected from Taq ligase and AMP ligase.

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

According to the embodiments of the present disclosure, the heat-intolerant DNA ligase includes at least one selected from T4 DNA ligase, E. coli DNA Ligase, T3 DNA Ligase, and T7 DNA Ligase.

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

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.

According to the embodiments 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.

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

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

According to the embodiments 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.

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:

Using a single-stranded circular DNA library and/or a double-stranded circular DNA library with a sequencing adapter sequence as a template, 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.

The above-mentioned nucleic acid amplification method is used to perform multi-copy amplification on the sequencing template, and the obtained single-stranded DNA multi-copy concatemer is loaded onto the sequencing chip as the base signal acquisition unit in sequencing, which has the following advantages: the DNA sequencing template of the DNA multi-copy concatemer has a large number of copies and a small volume, thereby occupying a small area of the sequencing chip or flow cell; the increase in the number of copies 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.

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.

According to an embodiment of the present disclosure, 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.

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

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

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 on the concatemer.

On the other hand, 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 strands, and the 5' end of the linear DNA single strand 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.

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 on the concatemer.

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.

In another aspect, the present disclosure provides a method for producing a second strand, the method 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.

According to an embodiment of the present disclosure, the primer is fixed 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.

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

The aforementioned single-stranded DNA multi-copy concatemer can be used as a base signal acquisition unit and serially loaded on a sequencing chip. The single-stranded DNA multi-copy concatemer is small in size, has a large number of copies, and a stable structure, which effectively solves the problems of difficult operation, loose structure, large size, and low amplification efficiency of DNA nanoballs obtained by rolling circle amplification. The aforementioned single-stranded DNA multi-copy concatemer is applied to the sequencing field, saving chip usage area, reducing costs, and improving accuracy.

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.

Additional aspects and advantages of the present disclosure will be given in part in the following description and in part will be obvious from the following description or learned through practice of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present disclosure will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

FIG1 shows a schematic flow chart of a nucleic acid amplification method according to an embodiment of the present disclosure;

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

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

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

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

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

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

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.

DETAILED DESCRIPTION OF THE INVENTION

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 construed as limiting the present disclosure.

In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

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 meanings commonly understood by those skilled in the art to which the present invention belongs.

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.

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.

Definitions and explanations of terms

In the present disclosure, the term "template" refers to the nucleic acid molecule to be tested, which means a polymer of nucleotides of a certain length, and the nucleotides 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.

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 may refer to the reading of any two segments or two parts that are not completely overlapping 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.

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

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

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.

In the present disclosure, the term "rolling circle amplification" is a newly 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 used to convert dNTPs into single-stranded DNA under enzyme catalysis. The single-stranded DNA contains hundreds or thousands of repeated template-complementary fragments.

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 an amplification and ligation reaction to form hundreds or thousands of copies of internally linked covalently linked circular DNA.

In the present disclosure, the term "DNA helicase" refers to a class of enzymes that unwind hydrogen bonds, and are enzymes that unwind DNA by hydrolyzing ATP to provide energy. They often rely on the presence of single strands and can recognize the single-stranded structure of replication forks. Generally, they play a role in catalyzing the unwinding of double-stranded DNA or RNA during DNA or RNA replication.

In the present disclosure, the term "single-strand binding protein", also known as DNA binding protein (SSB), 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, and a span of about 32 nucleotide units for binding to single-stranded DNA; after DNA unwinding, as long as the DNA molecules are base-paired, they tend to bind into double strands. 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. It does not move forward in the direction of replication like a polymerase, but instead constantly binds and detaches.

In the present disclosure, the term "exonuclease" refers to a type of enzyme that can sequentially catalyze the hydrolysis of 3, 5-phosphodiester bonds from one end of a polynucleotide chain to degrade nucleotides. The final product of its hydrolysis is a single nucleotide (dNMP for DNA and NMP for RNA). According to the difference in the characteristics of the action, it can be divided into single-stranded exonucleases and double-stranded exonucleases.

In the present disclosure, the term "Tm" value, melting temperature (Tm) refers to the temperature at which the absorbance value increases to half of the maximum value, which is called the melting temperature or melting point of DNA. The Tm value is the melting temperature of DNA, which is the temperature at which the ultraviolet absorption reaches half of the maximum value during the DNA denaturation process. DNA of different sequences has different Tm values. The higher the C-G content in the DNA, the greater the Tm value.

In the present disclosure, the term "base signal collection 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.

In the present disclosure, the term "strand displacement" refers to a chemically modified restriction site on a nucleic acid, which can open a gap in the DNA strand after being cleaved by the enzyme, and the DNA polymerase then extends the 3' end of the gap and replaces the next DNA strand. The replaced single-stranded DNA can bind to the primer and be extended into a double strand by the DNA polymerase. This process is repeated continuously, so that the nucleic acid sequence is efficiently amplified.

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

Nucleic Acid Amplification Methods

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

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

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

S130, performing an amplification and ligation reaction using the nucleic acid to be amplified and the primer pair to obtain a double-stranded DNA multi-copy concatemer.

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

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;

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 site;

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

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:

1) Extract total DNA from samples;

2) Use mechanical, ultrasonic or enzyme methods to break the DNA into fragments of different sizes, i.e. the DNA to be tested;

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

4) Designing a primer pair based on the linker, performing phosphorylation modification on the 5' ends of the two primers, and making at least one primer of the primer pair have a base modification;

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

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

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 the ligase to connect the 3' end of the chain amplification product to the 5' end of the primer to form a ring.

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

According to some embodiments of the present disclosure, the restriction site includes a ribonucleotide or deoxyribonucleotide that can be cut by the enzyme. The restriction 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 "restriction site" described in the present invention is not limited to the above types, and can also be other types of restriction sites. As long as the specific cutting of the enzyme can be achieved, this type of restriction site is included in the type of restriction site of the present invention.

According to some embodiments of the present disclosure, at least one of the primers in the primer pair carries a restriction site, which can be cut by an enzyme to form a gap on any one of the circular DNAs in the double-stranded DNA concatemer. For example, one of the primers in the primer pair carries a restriction site, which can be cut by an enzyme to cut the strand containing the restriction site in the double-stranded DNA concatemer to form a gap strand; or, the two primers in the primer pair contain different restriction sites, and the restriction sites of the double-stranded DNA concatemer are cut with corresponding enzymes as needed to form a gap strand.

According to some embodiments of the present disclosure, when the nucleic acid is a chain DNA, step (A) further comprises cyclizing the nucleic acid connected to the known sequence to obtain a cyclized nucleic acid to be amplified. In step (A) of the present invention, the nucleic acid to be amplified obtained after the nucleic acid is connected to the known sequence is preferably a cyclized 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.

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

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:

method one

The two primers for preparing the multi-copy DNA concatemer have different types of restriction sites, for example, primer 1 has uracil deoxyribonucleotides and primer 2 has ribonucleotides. When preparing a single-strand DNA multi-copy concatemer, after completing the amplification reaction of the amplification connection, RNase H is used to remove the ribonucleotide of primer 2, so that the chain amplified by primer 2 has a gap, and then it is removed by DNA digestion enzyme to obtain the first single-strand DNA multi-copy concatemer. After completing the sequencing of one chain, the complementary sequence generated during the sequencing process is eluted. Amplification is performed with primer 2, and a connection reaction is performed after one round of amplification to generate a two-strand DNA multi-copy concatemer. Then, the USER enzyme is used to cut the first chain to form a gap, and then the DNA digestion enzyme is used to remove the entire one-strand DNA multi-copy concatemer. Finally, a two-strand DNA multi-copy concatemer in a single-stranded state is obtained.

Method Two

After step (C), enzyme 2 is used to cut the restriction 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.

Method 3

The multi-copy concatemer of DNA obtained in step (c) or (D) is used as a template, and a primer complementary to the universal sequence of the first single-stranded DNA multi-copy concatemer linker is used to amplify 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.

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.

According to the embodiments of the present disclosure, the amplification and ligation reaction system contains a DNA polymerase, and the DNA polymerase 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 amplification product from being cleaved by enzymes.

According to the embodiments 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.

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

According to an embodiment of the present disclosure, the high temperature resistant DNA ligase includes at least one selected from Taq ligase and AMP ligase.

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

According to the embodiments of the present disclosure, the heat-intolerant DNA ligase is selected from at least one of T4 DNA ligase, E. coli DNA Ligase, T3 DNA Ligase, and T7 DNA Ligase.

According to the embodiments 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-stranded binding protein. The single-stranded binding protein contained in the system can prevent the unwound DNA from self-pairing.

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.

According to the embodiments 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.

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

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

According to the embodiments 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.

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

1) Use a single-stranded circular DNA library with a known linker sequence or a double-stranded circular DNA library as a template. The amplification primers are designed based on the known linker, and the two amplification primers may have overlapping complementary regions (the Tm value of the complementary fragment is lower than the Tm value of the two primers), or there may be no overlapping complementary regions. The 5' end of the primer is phosphorylated, and one of the primers includes a modified nucleotide that can be subsequently removed by the corresponding enzyme, such as uracil deoxyribonucleotide or ribonucleotide.

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. In addition to the template, the reaction system includes amplification primers, DNA polymerase, DNA ligase, dNTP, Mg ²⁺ ions, etc., wherein the DNA polymerase does not have 5'-3' exonuclease activity, such as KAPA HiFi HotStart DNA Polymerase or KAPA HiFi HotStart Uracil+Polymerase, and the DNA ligase can be a thermostable ligase, such as Taq ligase, AMPligase. It can also be a thermostable ligase, such as T4 DNA ligase. When a thermostable ligase is used, an isothermal amplification system is used, which may include DNA helicase, single-stranded binding protein, etc.

3) The template reacts in the amplification and ligation reaction system, and goes through the process of 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 be performed at the same temperature. DNA helicase can be used to achieve the effect of DNA denaturation and unwinding. After the primer is annealed to the template, the DNA polymerase extends and stops when it extends to the 5' end of the primer. The DNA ligase connects the 3' end of the extended chain with the 5' end of the primer itself to form a covalent double-stranded circular DNA. The two chains of the covalent double-stranded circular DNA are interlinked, and even if denaturation is performed, the two chains cannot be separated, and the two chains will remain entangled. When the cyclic amplification and ligation are performed again, all the products are interlinked and will not separate, and finally a double-stranded DNA multi-copy concatemer from the same template is obtained.

4) The obtained double-stranded multi-copy DNA concatemer product is digested to obtain a single-stranded DNA multi-copy concatemer. That is, a series of digestive enzymes are used to remove modifications such as uracil deoxyribonucleotides or ribonucleotides on one of the primers, such as using USER enzyme to remove U bases and using RNase H to remove ribonucleotides. A gap is created in one of the double-stranded DNA chains, and then a single-stranded DNA digestive enzyme, such as DNA exonuclease I and DNA exonuclease III, is used to digest the gapped single chain. These digestion reactions can exist in the same reaction system or in different reactions. Purification of the digested product can obtain a single-stranded DNA multi-copy concatemer.

According to some specific embodiments of the present disclosure, the present disclosure proposes a method for producing a second chain, which includes using a 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 chain.

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

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

Application in sequencing

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

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

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

method one

Using the current single-strand DNA multi-copy concatemer as a template, the adapter as a 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.

Method Two

The two primers for preparing the multi-copy DNA concatemer have different types of restriction sites, for example, primer 1 has uracil deoxyribonucleotides and primer 2 has ribonucleotides. When preparing a single-strand DNA multi-copy concatemer, after completing the amplification reaction of the amplification connection, RNase H is used to remove the ribonucleotide of primer 2, so that the chain amplified by primer 2 has a gap, and then the DNA digestion enzyme is used to remove it to obtain a single-stranded loop DNA multi-copy concatemer. After completing the sequencing of one chain, the complementary sequence generated during the sequencing process is eluted. Amplification is performed with primer 2, and a ligation reaction is performed after one circle of amplification to generate a two-strand DNA multi-copy concatemer. Then the USER enzyme is used to cut the one chain to form a gap, and then the DNA digestion enzyme is used to remove the entire one-strand DNA multi-copy concatemer. Finally, a two-strand DNA multi-copy concatemer in a single-stranded state is obtained.

The primer 1 and the primer 2 can have different restriction sites in any order, and in addition to U base and ribonucleic acid base, they can also be 5,6-dihydroxythymine, 5-hydroxyuracil, 5-hydroxymethyluracil, and 5-formyluracil, etc. The enzyme 1 and the enzyme 2 include at least one selected from USER enzyme, RNase H, UDG, RNase HII, and hSMUG1, and the enzyme 1 and the enzyme 2 are different enzymes.

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, and has the following advantages: the DNA sequencing template of the DNA multi-copy concatemer has a large number of copies and a small volume, thereby occupying a small area of the sequencing chip or the flow tank; the increase in the number of copies of the DNA sequencing template will not affect its volume, and the fragment length of the DNA sequencing template has a small 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 react on the sequencing chip or the flow tank, has low requirements for the chip, and does not need to generate nanowells on the chip and pre-plant oligonucleotides on the chip.

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

If no specific technology or conditions are specified in the implementation plan, the technology or conditions described in the literature in this field or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

Example 1 Escherichia coli genomic DNA sequencing

The experimental material in the embodiment is Escherichia coli genomic DNA

1. Experimental Procedure

(1) Prepare MGIEasy DNA Library Preparation Kit (16RXN) (MGI, Catalog No. 1000006987);

(2) Take 100 ng of E. coli genomic DNA, and according to the kit instructions, shear the DNA to a main band of 300 to 500 bp in size, and then undergo magnetic bead purification, fragment selection, end repair and A addition, adapter ligation, and PCR amplification. Finally, use the reagents in the single-stranded circularization part of the kit to circularize the PCR amplification product to obtain the final single-stranded circular DNA library suitable for the DNBSEQ sequencing platform;

(3) Take 5 ng of the prepared single-stranded circular DNA library into a 200 μL PCR tube;

(4) Prepare the amplification and ligation reaction solution on an ice box according to Table 1;

Table 1

Primer1:5’P-GCCATGTCGTTCTGTGAGCCAAGG-3’(SEQ ID NO：1)

Primer2: 5’P-GCTCACAGAA/rC//rG//rA//rC/ATGGCTACGATCCGAC-3’ (SEQ ID NO: 2)

Where r represents the modification of ribonucleotide and P represents phosphorylation modification.

(5) Add the prepared reaction solution to the PCR tube containing the single-stranded circular DNA library, and then add nuclease-free water (ThermoFisher, AM9930) to a final volume of 50 μL. Use an oscillating mixer to mix thoroughly, then centrifuge briefly to centrifuge the liquid to the bottom of the tube;

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

Table 2

(7) After the reaction is completed, take out the PCR tube, centrifuge it briefly, and place it on an ice box;

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

table 3

(9) Add the prepared digestion enzyme reaction solution to the PCR tube from the previous step. Use an oscillating mixer to mix thoroughly, then centrifuge briefly to centrifuge the liquid to the bottom of the tube;

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

Table 4

temperature time 45℃ hot cover On 37℃ 30min 4℃ Hold

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

(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.

(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.

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

(15) Repeat the previous step;

(16) Try to drain the liquid in the tube. If there is a small amount of liquid left on the tube wall, centrifuge the 1.5 mL centrifuge tube for a short time. After separation on the magnetic rack, use a small-range pipette to drain the liquid at the bottom of the tube.

(17) Keep the 1.5 mL centrifuge tube fixed on the magnetic rack, open the cap of the 1.5 mL centrifuge tube, and dry it at room temperature until the surface of the magnetic beads is not reflective and has no cracks;

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

(19) Incubate at room temperature for 5 min;

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

ssDNA Assay Kit荧光定量试剂盒，按照定量试剂盒的操作说明对酶切消化纯化后产物进行定量； (21) Use

ssDNA Assay Kit fluorescent quantitative kit, according to the operating instructions of the quantitative kit to quantify the product after enzyme digestion and purification;

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

(23) Prepare MGISEQ-2000 high-throughput sequencing reagent set (SE100) (MGI, 1000012551) and operate according to the instructions;

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

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

2. Experimental results

(1) After the single-stranded DNA sequencing library was reacted, the concentration of the DNA multi-copy concatemers was consistent with the expected concentration (Table 5);

Table 5 Input amount of single-stranded DNA library and DNC yield

(2) The electrophoresis diagram (Figure 6) showed that the band of the obtained product was too large (about ∼160 k) and did not run out of the spotting well, which was in line with expectations;

(3) After sequencing using DNBSEQ sequencing reagents and chips, a read length of more than 415M was obtained, and the Q30 quality value was more than 89%, achieving the expected goal;

(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).

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

In this embodiment, the whole process from obtaining the nucleic acid template to obtaining the amplified product (single-stranded DNA multi-copy concatemer) is simple to operate, and the required instruments and consumables are also conveniently available to experimental personnel in the field, which improves the experimental efficiency and reduces the experimental cost. It can be seen from Table 1 that a large amount of DNC can be obtained with the investment of a small amount of DNA library, and it can be seen from Figure 6 that the quality of the obtained DNC meets the standard, so this method can efficiently amplify the nucleic acid template while ensuring the quality of the amplified product. It can be seen from Figures 7 and 8 that in sequencing, the method disclosed in the present invention can significantly reduce the chip occupied area. Compared with the prior art, under the chip of the same area, the sequencing read length can be effectively improved, and because the template amplification is not performed on the chip, the chip requirements are relatively low.

Example 2 Sequencing of EGFR gene tumor hotspot regions

The experimental material in the embodiment is human genomic DNA

1. Experimental Procedure

(1) Design a primer pool for amplifying the tumor hotspot region of the EGFR gene, which contains 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.

Table 6

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;

(2) Take 5 ng of human genomic DNA into a PCR tube and perform PCR reaction according to the PCR reaction system configured as shown in (Table 7). The reaction procedure is as follows: 94℃ for 1 min; 94℃ for 30 s, 58℃ for 2 min, 72℃ for 30 s, 15 cycles; 72℃ for 5 min; 4℃∞.

Table 7

(3) After the reaction, the DNA was purified using the magnetic beads in the MGIEasy DNA Library Preparation Reagent Kit (16RXN) (MGI, Catalog No. 1000006987). Finally, the purified product was dissolved in 22 μL of elution buffer to obtain the PCR product.

(4) Take 300 ng of PCR amplification product and use the single-stranded circularization reagent in the MGIEasy DNA Library Preparation Reagent Set (16RXN) (MGI, Catalog No. 1000006987) to perform single-stranded circularization to obtain a single-stranded circular DNA library suitable for the DNBSEQ sequencing platform;

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

(6) Prepare the reaction solution for amplification and ligation reaction on an ice box according to Table 8;

Table 8

Primer1:5’P-GCCATGTCGTTCUGUGAGCCAAGG-3’(SEQ ID NO:19)

Primer2:5’P-GCTCACAGAA/rC//rG//rA//rC/ATGGCTACGATCCGAC-3’(SEQ ID NO:20)

Among them, r represents the modification of ribonucleotide; U represents deoxyribonucleic acid; and P represents phosphorylation modification.

(7) Add the prepared reaction solution to the PCR tube containing the single-stranded circular DNA library, and then add nuclease-free water (ThermoFisher, AM9930) to a final volume of 50 μL. Use an oscillating mixer to mix thoroughly, then centrifuge briefly to centrifuge the liquid to the bottom of the tube;

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

Table 9

(9) After the reaction is completed, take out the PCR tube, centrifuge it briefly, and place it on an ice box;

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

Table 10

(11) Add the prepared digestion enzyme reaction solution to the PCR tube from the previous step. Use an oscillating mixer to mix thoroughly, then centrifuge briefly to centrifuge the liquid to the bottom of the tube;

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

Table 11

temperature time 45℃ hot cover On 37℃ 30min 4℃ Hold

(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;

(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.

(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.

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

(17) Repeat the previous step;

(18) Try to drain the liquid in the tube. If there is a small amount of liquid left on the tube wall, centrifuge the 1.5 mL centrifuge tube for a short time. After separation on the magnetic rack, use a small-range pipette to drain the liquid at the bottom of the tube.

(19) Keep the 1.5 mL centrifuge tube fixed on the magnetic rack, open the cap of the 1.5 mL centrifuge tube, and dry it at room temperature until the surface of the magnetic beads is not reflective and has no cracks;

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

(21) Incubate at room temperature for 5 min;

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

ssDNA Assay Kit荧光定量试剂盒，按照定量试剂盒的操作说明对酶切消化纯化后产物进行定量； (23) Use

ssDNA Assay Kit fluorescent quantitative kit, according to the operating instructions of the quantitative kit to quantify the product after enzyme digestion and purification;

(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.

(25) Then, the reagents in wells 5 and 7 of the reagent reservoir were aspirated, and after washing twice with molecular-grade water, 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.

Primer for synthesizing the second strand: 5’P-GCTCACAGAACGACATGGCTACGATCCGAC-3’ (SEQ ID NO: 21)

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

Table 12

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

Table 13

(27) During the loading process, only the DNB loading buffer II in the kit was used, and the corresponding volume was 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.

(28) After completing the sequencing of the first strand, pump the formamide reagent into well 5, incubate at 85°C for 10 min, and then pump the cleaning reagent into well 5 for washing twice; then pump the primer for synthesizing the second strand into well 7, incubate at 65°C for 2 min, and then pump the cleaning reagent into well 7 for washing twice; then pump the enzyme reaction solution reagent for synthesizing the second strand into well 15, incubate at 65°C for 10 min, 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 min, and finally pump the cleaning reagent into well 15 for washing twice, thereby completing the synthesis of the second strand.

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

(30) After sequencing was completed, the obtained sequencing data were statistically analyzed. The data came 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 data of the 4M read length sequence were extracted for analysis and aligned to the human reference genome (hg19) using BWA software. Samtools was used to perform statistics on the data utilization, alignment rate, and target region data ratio of the sequencing data.

2. Experimental results

(1) After the single-stranded DNA sequencing library was reacted, the concentration of the DNA multi-copy concatemers was consistent with the expected concentration (Table 14);

Table 14 Input amount of single-stranded DNA library and DNC yield

(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 strand was more than 89%, and the Q30 quality value of the second strand was more than 80%, achieving the expected goal;

(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.

Table 15

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

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

Claims (20)

A nucleic acid amplification method, comprising: (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. A nucleic acid amplification method, comprising: (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. The method according to claim 1 or 2, 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. The nucleic acid amplification method according to claim 3, characterized in that step (c) or (c) further comprises: (D) When any one of the primers in the primer pair contains a restriction site 1 and the other primer does not contain a 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. The method according to any one of claims 1 to 4, characterized in that the 5' ends of the primers in the primer pair are all phosphorylated. The method according to any one of claims 1 to 5, characterized in that the restriction site comprises a modified ribonucleotide or deoxyribonucleotide that can be cleaved by the enzyme. The method according to any one of claims 1 to 6, characterized in that the restriction site comprises at least one selected from ribonucleotides, uracil deoxyribonucleotides, 5,6-dihydroxythymine deoxyribonucleotides, 5-hydroxyuracil deoxyribonucleotides, 5-hydroxymethyluracil deoxyribonucleotides, and 5-formyluracil deoxyribonucleotides. The method according to claim 1 or 2, characterized in that, when the nucleic acid is a chain DNA, step (A) or (a) further comprises subjecting the nucleic acid connected to the known sequence to a cyclization reaction to obtain a cyclized nucleic acid to be amplified. The method according to any one of claims 4 to 8, characterized in that the two primers in the primer pair contain restriction site 1 and restriction site 2, respectively, and the restriction 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. The method according to any one of claims 4 to 8, 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. A single-stranded DNA multi-copy concatemer, 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. The single-stranded DNA multi-copy concatemer according to claim 11 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. The single-stranded DNA multi-copy concatemer according to claim 12, 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. A nucleic acid composite molecule, characterized in that the nucleic acid composite molecule comprises a single-stranded DNA multi-copy concatemer according to any one of claims 11 to 13 and a plurality of linear DNA single strands, 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. The nucleic acid complex molecule according to claim 14 is characterized in that the 5' end of the linear DNA single strand is free and the 3' end is hybridized on the concatemer. 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. A method for generating a double strand, characterized in that the single-stranded DNA multi-copy concatemer described in 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 double strand. The method for generating a double strand according to claim 17, wherein the primer is fixed on a solid support. The method for generating a double strand according to claim 18, wherein the solid support may include a material selected from the group consisting of magnetic beads, polymers, hydrogels, metals, and chips. The method for generating a double strand according to any one of claims 17 to 19, wherein the primer is free in the solution.

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