KR20220161754A - Method for amplifying target dna - Google Patents

Abstract

The present application relates to a method for amplifying molecularly-marked target DNA, and a method for sequencing target DNA. More specifically, the present invention relates to: a method for amplifying desired target DNA from complex DNA samples such as genomes; and a target DNA sequencing method using the same. The present invention can be usefully used to determine an accurate target DNA sequence without errors by constructing a common sequence of sequences in which molecular markers are the same.

Description

Target DNA amplification method {METHOD FOR AMPLIFYING TARGET DNA}

The present application relates to a molecular-labeled target DNA amplification method and a target DNA sequencing method, and more particularly, to a method for amplifying a desired target DNA from a complex DNA sample such as a genome and a target DNA sequencing method using the same.

Detection of DNA (deoxyribonucleic acid) having a specific nucleotide sequence, that is, target DNA (target DNA), can be applied to the diagnosis of pathogenic or hereditary diseases, and thus has attracted much attention.

When nucleic acids are extracted from biological samples such as blood for analysis of nucleic acids (DNA or RNA) in living organisms, the amount extracted is generally too small to be directly used for various analyses, so it is necessary to amplify the extracted nucleic acids to accurately analyze them. do. Various methods of nucleic acid amplification technology have been developed to date. In particular, PCR (Polymerase Chain Reaction), a representative method for amplifying DNA, is a high-efficiency amplification technology that selectively amplifies target genes in large quantities. Under this background, the present inventors have made diligent efforts to develop a method that can effectively and accurately analyze amplified target nucleic acids compared to conventional methods, and as a result, target DNA using molecularly labeled circular DNA It was confirmed that it could be effectively amplified, and this application was completed.

One object of the present application is, (1) a first step of coupling a double-stranded adapter having a cleavage surface and complementary ends of the cleaved DNA; (2) a second step of amplifying the DNA to which the adapter is attached; (3) a third step of manipulating the terminal sequence to enable ligation for circularization of the amplified DNA; (4) a fourth step of circularizing the DNA in the third step; and (5) a fifth step of amplifying a target sequence region from the circularized DNA prepared in the fourth step.

Another object of the present application is to provide a target DNA sequencing method comprising the steps of grouping amplification products prepared by the method according to templates and constructing a common sequence for each group.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the present application described herein. Also, such equivalents are intended to be included in this application.

In order to achieve the above object, one aspect of the present invention,

(1) For DNA extracted from cells, tissues, or bodily fluids such as blood, or DNA in a state where additional cleavage has been performed through physical or chemical methods, the ends are arranged such as A-tailing. A first step of coupling with the next prepared adapter;

(2) Using primers that provide adapter-specific sequences for the DNA to which adapters are bound at both ends, and additional structures that allow DNA products generated after PCR amplification to cleave specific sites of adapter sites by cleaving enzymes. A second step of amplification;

(3) The third step of preparing DNA circularization by cutting the terminal part of the DNA amplified in the second step with a restriction enzyme that recognizes a specific sequence or structure and, if necessary, by limiting the modification of the terminal structure through DNA polymerization.

(4) The 4th step of circularizing the DNA prepared in the 3rd step through self-ligation or ligation using an adapter having a structure complementary to the terminal part.

(5) A target using molecularly labeled circularized DNA comprising a fifth step of amplifying the target sequence using the DNA circularized in the fourth step as a template and using a primer pair specific to the target sequence A method for amplifying DNA is provided.

The first to fifth steps may be provided as steps for preparing a library for Next Generation Sequencing (NGS).

In the present invention, the term "next generation sequencing (NGS)" refers to a high-speed analysis method for genome sequencing, and may be used interchangeably with high-throughput sequencing, massive parallel sequencing, or second generation sequencing.

In the present invention, the term "library" is a collection of DNA fragments having common characteristics genetically or structurally or digested with restriction enzymes, etc., prepared to be sequenced through NGS, but is not limited thereto. Specifically, in the present invention, the library can be prepared through the first to fifth steps.

The first step provides a step of combining DNA extracted from various samples with an adapter having a specific structure.

In the present invention, the term "adapter" refers to a base sequence of a double helix structure used to amplify a DNA sequence located between adapters using primers having adapter-specific sequences by binding to both ends of a DNA fragment, and partially It may have a single-stranded region and a portion of the double-stranded region may consist of non-complementary base pairs.

The adapter includes complementary binding of two oligonucleotides, Top_oligo and Bot_oligo, followed by a fill-in process of filling the single-stranded region with complementary base pairs, and restriction enzyme cleavage to create a 3'-T overhang structure on one side of the terminal It may consist of a process (Fig. 1).

At this time, Top and Bot, which represent oligos, point to the top and bottom to make it easy to distinguish the two strands when the double-stranded DNA, which is a pair of complementary base sequences, is pictured, and does not actually indicate up and down.

The adapter may be composed of a restriction enzyme recognition site, a molecular marker site and a sequence site, but is not limited thereto.

Specifically, Top_oilgo for constructing the adapter contains a restriction enzyme recognition site at the 5' side of the sequence that can form a 3'-T overhang at the end when the restriction enzyme Hpy188I is treated on a double-stranded structure, and below it It consists of a molecular marker region containing several Ns and a sequence region capable of complementary binding with Bot_oligo. At this time, N constituting the molecular marker makes bases A, G, T, and C randomly appear at the position occupied by N on the sequence, and forms various sequence combinations through several base configurations represented by N. DNA bound to the adapter It provides a means to differentiate the pieces. In addition, non-complementary base pairs can be placed at the complementary binding sites of the two oligos to distinguish each strand of the duplex for the DNA fragment binding to the adapter.

The step of binding the adapter of the present invention may include, but is not limited to, a step of performing fill-in of a single strand of the adapter end using a DNA polymerase.

Specifically, their complementary bonding is completed by raising the temperature of a solution in which the two oligos are dissolved at the same molar ratio to 95° C. and then slowly cooling them down. After the two oligos form complementary bonds, the single-stranded region forms a complete double-stranded structure by performing a fill-in reaction using DNA polymerase and dNTP as substrates. To bind the adapter to the A-tailed DNA, the double-stranded adapter is digested with Hpy188I restriction enzyme to create a 3'-T overhang at one end, thereby completing the adapter (Fig. 2).

DNA fragments to be bound to adapters can be collected from samples in various forms or states, such as tissues, cells, and body fluids. Further cleavage can be cleaved to a certain size range using a physical method using ultrasound, or can be cleaved using an enzymatic system composed of several endonuclease cocktails.

If necessary, trimming, fill-in, etc. are performed on both ends of the cut DNA, followed by A-tailing, which follows the usual method. DNA fragments 3' A-tailed at both ends are ligated using the above adapter and DNA ligase to complete the first step (Fig. 3).

The second step provides a step of amplifying the adapter-linked DNAs through PCR.

The primer is designed to bind to the outside of the non-complementary base pair of the adapter site, and when the DNA to which the adapter is bound is amplified, the site where the non-complementary base pair is located in each amplification product base sequence of the double-stranded DNA appears differently from each other, so that each amplification product is doubled. Make sure you know which side of the strand it came from.

In addition, the primers have sequences specific to the adapter DNA and may include structural devices in the primers to form end structures for DNA circularization (Figure 4).

Specifically, in the adapter-specific sequence, as shown in FIG. 3, methyl-cytosine was used at the site indicated by X in the primer sequence, which together with neighboring cytosine provides a recognition site for the FspE1 restriction enzyme.

The third step provides a step of processing the ends of the amplified DNA to form a structure that facilitates the creation of circularized DNA. FspE1 recognizes the CmC sequence structure present in the DNA sequence, cleaves the phosphor-diester bond 12 bases to the 3' side of the DNA strand with this recognition sequence, and 5' in the DNA strand with the complementary sequence of the CmC strand. By cleaving the phosphor-diester bond next to 16 bases to the side, a terminal having an overhang structure of 4 bases on the 5' side is generated (Fig. 5 and Fig. 6). At this time, since the FspEI restriction enzyme cleaves outside the recognition site, adapter sequences can be designed so that the overhangs of both ends after cleavage are complementary or non-complementary with each other, if necessary.

The fourth step provides a step of circularizing the amplified DNA with both ends processed. Circularization through autologous ligation using a 5' overhang by FspEI or circularization through ligation rebound with the insertion of a small adapter is provided. At this time, the adapter for circularization has overhangs complementary to the overhangs formed at both ends. In addition, even if only one strand of the adapter is circularized through ligation, a phosphate group can be placed at the 5' end of one strand of the adapter. When both strands are circular, the physical pressure in the direction of polymerization increases due to the twisted structure of the circularized DNA during the PCR amplification process in step 5, which can inhibit the polymerization reaction. can be resolved (Figs. 7, 8 and 9)

The fifth step provides a step of amplifying the target sequence in a form containing a molecular marker using a primer pair specific to the target sequence (FIGS. 9 and 10).

Specifically, PCR may be performed using the product prepared in the fourth step as a template and using a pair of primers binding to both ends of the template, but is not limited thereto.

In the present invention, the term "amplification product" means a result of PCR performed using primers, and may be used in combination with amplified DNA.

The amplified DNA includes different labels according to each template to be amplified.

The fifth step may be performed on circularized DNA, but is not limited thereto.

Specifically, the direction of the primers is designed so that each primer faces the molecular marker in the circularized DNA structure. Since this form proceeds in the direction where they do not meet each other in the non-circularized state, amplification does not occur.

After determining the nucleotide sequence of the DNA amplified in the fifth step, sequences having the same template origin can be collected in consideration of the sequence of the molecular marker inserted in the middle and the relative insertion position. By constructing a common consensus sequence from these collections, it provides a means to determine the exact base sequence of the target DNA. If there are multiple target sequences to be amplified, primer pairs for each target can be prepared in the same manner as above and multiple targets can be amplified to prepare a library for target DNA.

After the fifth step, an NGS process may be additionally performed.

Another aspect of the present application provides an adapter having a specific structure composed of a restriction enzyme recognition site, a molecular marker site, and a sequence site.

The adapter of the present application is as described above.

Another aspect of the present application is,

(1) For DNA extracted from cells, tissues, or bodily fluids such as blood, or DNA in a state where additional cleavage has been performed through physical or chemical methods, the ends are arranged such as A-tailing. A first step of coupling with the next prepared adapter;

(2) Using primers that provide adapter-specific sequences for the DNA to which adapters are bound at both ends, and additional structures that allow DNA products generated after PCR amplification to cleave specific sites of adapter sites by cleaving enzymes. A second step of amplification;

(3) The third step of preparing DNA circularization by cutting the terminal part of the DNA amplified in the second step with a restriction enzyme that recognizes a specific sequence or structure and, if necessary, by limiting the modification of the terminal structure through DNA polymerization.

(4) Provides a method for producing circularized molecularly labeled DNA comprising a fourth step of circularizing the DNA prepared in the third step through self-ligation or ligation using an adapter having a structure complementary to the end portion.

Another aspect of the present application provides a target DNA sequencing method comprising grouping amplification products prepared by the method according to templates and constructing a common sequence for each group.

The amplification products of the present application are as described above.

The amplified DNA contains different labels according to each template to be amplified. Therefore, each sequence can be grouped by template through sequencing of the amplified DNA, and an accurate target DNA sequence can be determined by constructing a common sequence for each group.

Various biochemical reactions involved in the sequencing process and the sequencing reaction itself include a significant level of errors in the determined base sequence, and these errors make it difficult to accurately detect or measure low-frequency mutations included in the genome.

Since the present invention can effectively eliminate the above-mentioned errors by constructing a common sequence of identical sequences with molecular markers, it can be effectively provided to determine the type, number, scale, etc. of mutations included in a genome target sequence.

The target DNA amplification method and the target DNA sequencing method of the present application can amplify a desired target DNA from a complex DNA sample such as a genome using molecularly labeled circularized DNA. In addition, since the amplified DNA includes different labels according to each template to be amplified, molecular markers can be usefully used to determine an accurate target DNA sequence without error by constructing a common sequence of the same sequences.

1 shows the structure and description of the adapters Top_oligo and Bot_oligo prepared in Example.
Figure 2 shows the manufacturing process of the adapter.
3 shows that adapters are bound to both ends of the A-tailed DNA fragment, and each strand is shown in different colors because the sequences of the adapter strands bound to the two strands of the DNA fragment are different.
Figure 4 shows the primer sequence for amplifying the DNA fragment to which the adapter is bound.
FIG. 5 shows a form in which each strand of the DNA fragment is amplified when the adapter-linked DNA fragment is amplified using the primers shown in FIG.
Figure 6 shows the structure of both ends of the DNA when the amplified DNA is digested with FspEI.
FIG. 7 shows an oligo constituting an adapter to be inserted when the DNA of FIG. 6 is circularized.
Figure 8 shows an adapter structure prepared using the oligo of Figure 7.
Figure 9 shows circularization by inserting the adapter of Figure 8 into the cut DNA of Figure 6.
FIG. 10 shows structures of the target sequence and an artificial sequence included therebetween when the target sequence is amplified using the circularized DNA of FIG. 9 as a template.
FIG. 11 shows the electrophoresis of the DNA to which the adapter of FIG. 3 was attached by PCR amplification using the primers of FIG. 4 on an agarose gel.
FIG. 12 shows the result of electrophoresis on an agarose gel by amplifying two target DNAs, respectively, using the DNA subjected to ligation for insertion of the CAST adapter and end-truncation of the amplified DNA of FIG. 11 as a template.
FIG. 13 shows the results of simultaneous amplification of multiple target DNAs and electrophoresis on an agarose gel using the DNA subjected to ligation for insertion of the CAST adapter and end-truncation of the amplified DNA of FIG. 11 as a template.

Hereinafter, the present application will be described in more detail through examples. However, these examples are intended to illustrate the present application by way of example, and the scope of the present application is not limited to these examples.

Example 1: Fabrication of adapter

As shown in the structure shown in Figure 1, oligonucleotides for preparing adapters were prepared. Specifically, Top_oligo was included to include Hpy188I restriction enzyme recognition sequence site 1, molecular marker site 2, and site 3 that is cleaved by FspE1 when the DNA to which the adapter is coupled is amplified using the primers shown in FIG. 4 . Bot_oligo has a complementary sequence with the 3'-side of Top_Oligo, but each strand of DNA to which this adapter is bound can be distinguished by having a non-complementary base pair on the 4th part of Bot_oligo that binds to the 3rd part of Top_oligo. At this time, the characteristic site and sequence of each oligo are exemplified to explain the role of each oligonucleotide, and the base sequence and position of the characteristic site within the ring may vary within the range where each role is not affected.

As shown in FIG. 2, after complementary coupling of the two oligos, klenow enzyme was used to fill-in the single-stranded region of Top_oligo with complementary base pairs. A partially double-stranded adapter having each cleaved surface and complementary ends was prepared so that one side of the DNA has a 3'-T overhang by treating the double-stranded DNA filled-in with Hpy188I restriction enzyme. This structure is to make it easy to bind the adapter to the DNA to be amplified by binding the adapter to the terminal structure having a 3'-A tail, and depending on the structure of the terminal part of the DNA to be bound, Restriction enzymes can be different. Adapters were prepared by purifying the cut DNA.

Top_oligo and Bot_oligo, which constitute the adapter, were produced by commissioning to Bioneer Co., Ltd. Using the above two oligonucleotides, equal amounts of 50 μl were mixed at a concentration of 100 pmole/μl, respectively. Then, after leaving it at 95 ° C for 5 minutes and at 75 ° C for 5 minutes in turn, the temperature was lowered from 75 ° C to 35 ° C at a rate of 1 ° C / cycle / min to induce complementary bonding between the two base sequences, thereby partially double-stranded. DNA was made. 5 units of klenow enzyme, 3 μl of buffer, 0.6 μl of 10 mM dDNT, and 5.4 μl of distilled water were added to 20 μl of partial double-stranded DNA, followed by a fill-in reaction at 37° C. for 30 minutes. Fill-in double-stranded DNA was purified using a DNA purification kit and then dissolved in 40 μl of elution buffer. After adding 10 units of restriction enzyme Hpy188I, 5 μl of buffer, and 4 μl of water, the mixture was reacted at 37°C for 4 hours, and purified using a DNA purification kit to prepare an adapter.

Example 2: Cleavage of Sample DNA and Adapter Binding and Amplification

Genomic DNA extracted from human cell lines was cut to a length of 300-500 bp using Cobaris. The cut DNA was subjected to end polish and 3'-A tailing. End-polish makes both ends of DNA blunt ends and attaches phosphate to 5'-end. 3 units of T4 DNA polymerase, 10 units of T4 polynucelotide kinase, 10 mM dNTP 1 ul, 10 2.5 ul of mM ATP and 2.5 ul of buffer were added and reacted at 25°C for 20 minutes. End polished DNA was purified with a DNA purification kit, only 1μl of 10 mM dATP was added as a substrate, and 3'-A tailing was performed by reacting at 37℃ for 30 minutes using 1 unit of Taq polymerase. The 3'-end A-tailed cleaved DNA in the reaction solution was purified using a DNA purification kit.

40 ng of 3'-end A-tailed DNA was cleaved with Blunt/TA ligase master mix (2X, NEB) at 25° C. for 30 minutes, and the adapter cut with Hpy1881 of FIG. 2 was reacted and ligated. Adapter-bound DNA was purified with a DNA purification kit.

10 ng of adapter-linked DNA was used as a template and amplified through PCR. At this time, the primer CAST4 of FIG. 4 specific to the adapter sequence was used. The PCR reaction was performed in a thermocyler after adding 0.4 μl of 10 mM dNTP and 2 units of h-Taq DNA polymerase to the template DNA under the following reaction conditions: 95° C. for 15 minutes once; 32 cycles of 95°C for 10 seconds, 57°C for 30 seconds, and 68°C for 40 seconds; 68 ℃ 10 minutes once. The amplified DNA was electrophoresed on a 2% agarose gel and is shown in FIG. 11 . The portion marked with X in the above primer sequence is a methylated cytosine and provides a recognition site for the restriction enzyme FspEI together with a neighboring cytosine. Therefore, all DNA amplified by this primer is cleaved by FspEI.

Example 3: Circularization of amplified DNA and single target DNA amplification

5 units of FspEI enzyme, 0.6 ul of enzyme activator solution, and 3 ul of buffer were added to 400 ng of the DNA amplified in Example 2, followed by reaction at 37°C for 4 hours to cleave the DNA end and purify with a DNA purification kit. Both ends of DNA have a 5'-overhang structure of 4 bases by enzymatic digestion, and the overhangs of the two ends are not complementary to each other. For circularization of this DNA, DNA circularization was performed by performing a ligation reaction using a small adapter DNA having a 4-nucleotide overhang complementary to each of the two ends. The CAST adapter of FIG. 8 was used by complementary coupling of the two oligos of FIG. 7 without fill-in (Ins4T/4B). To 50 ng of DNA digested with FspEI, CAST adapter and Blunt/TA ligase master mix were added, reacted at 25°C for 2 hours to circularize DNA, and purified with a DNA purification kit. PCR was performed using 10 ng of circularized DNA as a template and using a primer pair specific to the target DNA. At this time, PCR was performed on each of the two targets to selectively amplify the target DNA (FIG. 12). The PCR conditions were as follows: 15 minutes at 95° C.; 38 cycles of 95°C for 10 seconds, 57°C for 30 seconds, and 68°C for 40 seconds; 68 ℃ 10 minutes once. At this time, the amplified DNA includes molecular markers and adapter sequences in the middle of the amplification product.

Example 4: Circularization of amplified DNA and amplification of multiple target DNA

Target DNA was selectively amplified by performing PCR using 10 ng of the circularized DNA in Example 3 as a template and using multiple primer pairs specific to multiple target DNAs (FIG. 13). At this time, multiple PCR was performed under three conditions to include 10, 20, and 50 primer pairs, respectively. The PCR conditions were as follows: 1 time at 95° C. for 15 minutes; 38 cycles of 95°C for 10 seconds, 57°C for 3 minutes, and 68°C for 40 seconds; 68 ℃ 10 minutes once. At this time, the amplified DNA includes the molecular marker in the middle of the amplification product and the overhang sequence of the ligated DNA strand.

Example 5: NGS performance and sequencing of target DNA of amplified DNA

A sequencing library for NGS was prepared with the amplification products for the two target sites generated in Example 3, NGS was performed, and sequencing was performed. Whether the sequences produced from each library were derived from the target DNA and whether or not the molecular index was included was analyzed (Fig. 14). It was confirmed that an average of 91.5% of the sequences produced for each sample were derived from the target DNA, and it was found that molecular markers were included in 47% of the sequences.

A sequencing library for NGS was prepared with the amplification products for the multiple target sites generated in Example 4, NGS was performed, and sequencing was performed. Whether the sequences produced from each library were derived from the target DNA and included a molecular index were analyzed (Fig. 15). It was confirmed that an average of 44.2% of the sequences produced for each sample were derived from the target DNA. When the number of targets was varied, the magnitude of the number of targets amplified in each sample ranged from 86% to 100%.

From the above description, those skilled in the art to which this application pertains will be able to understand that this application can be implemented in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts rather than the detailed description above.

Claims (7)

A molecularly labeled target DNA amplification method comprising the following steps 1 to 5:
(1) a first step of linking the cleavage surface of the cleaved DNA with a double-stranded adapter having complementary ends;
(2) a second step of amplifying the DNA to which the adapter is attached;
(3) a third step of manipulating the terminal sequence to enable ligation for circularization of the amplified DNA;
(4) a fourth step of circularizing the DNA in the third step; and
(5) A fifth step of amplifying a target sequence region from the circularized DNA prepared in the fourth step.
The method of claim 1, wherein the adapter of the first step is composed of a restriction enzyme recognition site, a molecular marker site, and a sequence site.
According to claim 1,
The step of binding the adapter in the first step comprises performing fill-in of the adapter terminal single strand using a DNA polymerase.
According to claim 1,
Wherein the fifth step comprises performing PCR using the product prepared in the fourth step as a template and using a primer pair binding to both ends of the template.
According to claim 1,
Wherein the fifth step is made for the circularized DNA.
According to claim 1,
The method of further performing NGS (Next Generation Sequencing) after the fifth step.
Grouping amplification products prepared by the method according to any one of claims 1 to 5 by template; and constructing a common sequence of each group.

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