CN118147274A

CN118147274A - Fragmenting enzyme reaction system for reducing quantity of hairpin structures formed by DNA interruption and DNA library construction method

Info

Publication number: CN118147274A
Application number: CN202410337577.5A
Authority: CN
Inventors: 郭艳; 李迎迎; 王举梅; 刘玉方; 李晓晨; 孙克非
Original assignee: Tiangen Biotech Beijing Co Ltd
Current assignee: Tiangen Biotech Beijing Co Ltd
Priority date: 2024-03-23
Filing date: 2024-03-23
Publication date: 2024-06-07

Abstract

The application relates to the technical field of DNA interruption, and particularly discloses a fragmenting enzyme reaction system for reducing the number of hairpin structures formed by DNA interruption and a DNA library construction method. The fragmenting enzyme reaction system comprises: combining enzymes and breaking reactants; the combination enzyme comprises restriction enzyme and polymerase; the breaking reactants comprise Mg ²⁺ and Ca ²⁺, and are free of manganese ions; in the fragmenting enzyme reaction system, the concentration of Mg ²⁺ is 0.5-0.9 mM, and the concentration of Ca ²⁺ is 0.3-0.7 mM. The application reduces the frequency of DNA palindromic sequence exposure under the action of restriction endonuclease by screening the types and the concentrations of metal ions and auxiliary enzymes, thereby reducing the formation quantity of hairpin structures in the process of breaking DNA.

Description

Fragmenting enzyme reaction system for reducing quantity of hairpin structures formed by DNA interruption and DNA library construction method

Technical Field

The application relates to the technical field of DNA interruption, in particular to a fragmenting enzyme reaction system for reducing the number of hairpin structures formed by DNA interruption and a DNA library construction method.

Background

In NGS (next generation sequencing technology) experiments, the first step in DNA library construction is fragmentation of gDNA (genomic DNA), which randomly breaks the DNA into fragment sizes that fit sequencing. The most common breaking modes at present are physical breaking and enzymatic breaking, and both can efficiently fragment DNA, but the two methods have advantages in practical use and need to be selected according to specific experimental conditions. The physical breaking method mainly comprises ultrasonic or acoustic confocal (Covaris), and the shearing force generated by ultrasonic can shear DNA fragments in the solution, but the method needs special instruments, and has the problems of long service life, large gDNA input amount, low product recovery efficiency and the like. Enzymatic disruption is the random, nonspecific cleavage of DNA. The enzyme breaking method is convenient to operate, breaking, terminal repairing and A adding can be completed in one step, experimental steps are reduced, and experimental cost can be obviously reduced. The general enzyme cutting and breaking modes mainly comprise two modes: one is to create an incision with one endonuclease and then cut the sequence opposite the incision with the other endonuclease; another way of disruption is to randomly create nicks on both strands of DNA using one endonuclease and then fragment the DNA by strand displacement with another DNA polymerase.

However, according to Gregory T et al, DNA molecules are exposed to certain reverse complement sequences under the action of enzymes, the binding of which results in the formation of hairpin structures. Chigusa S et al demonstrated that the number of ARTIFACT SNV/Indel (artificial single nucleotide variant small fragment insertions) in the enzymatically constructed library was much greater than with the mechanical disruption method. Therefore, high-depth sequencing of tumor NGS detection brings a large number of false positives and brings a certain difficulty to interpretation of NGS results.

Disclosure of Invention

In view of the problem that more hairpin structures are generated in the current process of cutting and breaking DNA, the application provides a reagent and a method for a fragmenting enzyme reaction system, which can reduce the quantity of hairpin structures formed in the process of cutting and breaking DNA.

In a first aspect, the present application provides a reaction system for a fragmenting enzyme that reduces the number of hairpin structures formed by DNA disruption, and adopts the following technical scheme.

A fragmenting enzyme reaction system that reduces the number of hairpin structures formed by DNA breaks, the fragmenting enzyme reaction system comprising: combining enzymes and breaking reactants; the combination enzyme comprises restriction enzyme and polymerase; the breaking reactants comprise Mg ²⁺ and Ca ²⁺, and are free of manganese ions; in the reaction system of the fragmenting enzyme, the concentration of Mg ²⁺ is 0.5-0.9 mM, and the concentration of Ca ²⁺ is 0.3-0.7 mM.

By adopting the technical scheme, in the process of optimizing the reaction system of the fragmenting enzyme, metal ions and breaking auxiliary enzymes in the system can influence the exposure of palindromic sequences in the breaking process, wherein Mg ²⁺ and Ca ²⁺ can maintain the activities of the restriction enzymes and the polymerases to be higher, and manganese ions have a certain inhibition effect on the activities of the restriction enzymes and the polymerases. Restriction enzymes randomly generate nicks on two strands of DNA, and then fragment the DNA by strand displacement by polymerases. Through an optimization scheme, a fragmenting enzyme reaction system containing Mg ²⁺ with the concentration of 0.5-0.9 mM and Ca ²⁺ with the concentration of 0.3-0.7 mM is selected, so that the quantity of hairpin structures formed in the DNA breaking process can be reduced better.

As an improvement of the system for the reaction of the fragmenting enzyme which reduces the number of hairpin structures formed by DNA interruption, the combination enzyme also comprises a T4 single-chain DNA binding protein.

By adopting the technical scheme, the T4 single-stranded DNA binding protein is abbreviated as T4GP32, so that the restriction enzyme digestion reaction of the restriction enzyme on DNA can be promoted, the activity of polymerase can be enhanced, and the number of hairpin structures formed in the DNA breaking process can be reduced.

As an improvement of the fragmenting enzyme reaction system for reducing the number of hairpin structures formed by DNA interruption, the concentration of the T4 single-chain DNA binding protein in the fragmenting enzyme reaction system is 0.06-0.2 mug/mug.

By adopting the technical scheme, the number of hairpin structures formed by the breaking process of the fragmenting enzyme reaction system on DNA is smaller under the concentration of the T4 single-stranded DNA binding protein.

As an improvement to the system of the fragmenting enzyme reaction that reduces the number of hairpin structures formed by DNA fragmentation, the fragmentation reactants include TrisHCl, naCl, mgCl ₂、CaCl₂ and dNTPs; the concentration of each component of the breaking reactant in the fragmenting enzyme reaction system is as follows: the concentration of TrisHCl is 10-50 mM, the concentration of NaCl is 20-60 mM, the concentration of MgCl ₂ is 0.5-0.9 mM, the concentration of CaCl ₂ is 0.3-0.7 mM, and the concentration of dNTPs is 0.03-0.07 mM.

By adopting the technical scheme, the reaction system with the concentration is characterized in that the pH value of TrisHCl is regulated to 7.5-8.5, a stable pH environment is provided for the enzyme, the solubility of DNA is improved by NaCl, the activities of restriction enzymes and polymerases are maintained by MgCl ₂ and CaCl ₂, and the DNA is subjected to end repair by dNTPs under the action of the polymerases.

As an improvement of the system for the reaction of the fragmenting enzyme which reduces the number of hairpin structures formed by the DNA interruption, the restriction enzyme is selected from one or both of DNase I and dsDNase.

By adopting the technical scheme, DNase I and dsDNase randomly shear any site of double-stranded DNA under the condition of proper concentration of Mg ²⁺ and Ca ²⁺. dsDNase can cleave phosphodiester bonds in DNA to generate oligonucleotides with 5 '-phosphate and 3' -hydroxyl ends, effecting disruption of DNA.

As an improvement to the system for the reaction of the fragmenting enzyme which reduces the number of hairpin structures formed by the DNA interruption, the polymerase is selected from one or more of Klenow DNA polymerase, taq DNA polymerase and BST DNA polymerase.

By adopting the technical scheme, under the condition of proper concentration of Mg ²⁺ and Ca ²⁺, the polymerases can catalyze DNA strand displacement to fragment DNA, and simultaneously Klenow DNA polymerase has 5' -end cutting capability and 5' -3' polymerase activity of DNA, can catalyze DNA end repair and A, taq DNA polymerase and BST DNA polymerase both have 5 '. Fwdarw.3 ' exonuclease activity of strand displacement depending on DNA synthesis, and can catalyze DNA end repair and A.

As an improvement of the system for the reaction of the fragmenting enzyme which reduces the number of hairpin structures formed by the DNA disruption, the restriction enzyme is DNase I; the polymerase is a combination of Klenow DNA polymerase and Taq DNA polymerase; in the reaction system of the fragmenting enzyme: DNase I concentration is 0.00005-0.0005U/. Mu.L, klenow DNA polymerase concentration is 0.01-0.03U/. Mu.L, taq DNA polymerase concentration is 0.03-0.07U/. Mu.L.

By adopting the technical scheme, restriction enzymes and polymerases with the types and the concentrations can generate less hairpin structures in the process of breaking DNA under the auxiliary action of Mg ²⁺ and Ca ² ⁺.

In a second aspect, the present application provides a method for constructing a DNA library, and adopts the following technical scheme.

A method for constructing a DNA library, which utilizes the above-mentioned fragmenting enzyme reaction system to construct a DNA library; the DNA library construction method comprises the following steps:

s1, generating a notch on a DNA chain by using the restriction endonuclease, and then carrying out chain replacement by using the polymerase to fragment the DNA to obtain fragmented DNA; continuously carrying out terminal repair and 3' -terminal addition A on the fragmented DNA by using the polymerase to obtain a DNA addition A product;

s2, connecting the DNA addition product with a connector with a protruding T at the 5' end by using ligase to obtain a DNA connection product;

S3, purifying the DNA ligation product;

S4, carrying out PCR amplification by taking the purified DNA connection product as a template, and constructing to obtain a DNA library.

By adopting the technical scheme, the DNA library with a small proportion of hairpin structures is constructed.

As an improvement of the DNA library construction method, the reaction conditions of the step S1 are as follows: mixing the DNA sample with the reaction system of the fragmenting enzyme, keeping the temperature at 36-38 ℃ for 5-15 min, and then keeping the temperature at 60-70 ℃ for 20-40 min, so that the DNA is continuously fragmented, the tail end is repaired, and the 3' -end is added with A, and a DNA added A product is obtained.

By adopting the technical scheme, under the reaction conditions, DNA is continuously broken, terminal repair and A addition are carried out in the same reaction reagent through one-step reaction, and the hairpin structure formed by the DNA is less.

In summary, the method for constructing the DNA library and the system for reacting the DNA fragments to reduce the number of hairpin structures formed by DNA interruption have the following beneficial effects: the selection of the fragmenting enzyme reaction system containing Mg ²⁺ to 0.9mM, ca ²⁺ to 0.3 to 0.7mM, restriction enzymes and polymerase can preferably reduce the number of hairpin structures formed in the DNA breaking process. The T4 single-chain DNA binding protein is added into the fragmenting enzyme reaction system, so that the digestion reaction of restriction enzyme on DNA can be further promoted, the activity of polymerase can be enhanced, and the number of hairpin structures formed in the DNA breaking process can be further reduced. The application reduces the frequency of DNA palindromic sequence exposure under the action of restriction endonuclease by screening the types and the concentrations of metal ions and auxiliary enzymes, thereby reducing the number of hairpin structures formed in the process of breaking DNA.

Drawings

FIG. 1 is a 2100 peak plot of library 1 and library 2 of example 1 under a break buffer containing calcium ions, magnesium ions, and manganese ions.

FIG. 2 is a 2100 peak plot of library 3 and library 4 of example 2 under a break buffer containing calcium and magnesium ions.

FIG. 3 is a 2100 peak plot of libraries 5-7 at different concentrations of helper protein T4GP32 for the combination enzyme of example 3.

Detailed Description

The following describes the embodiments of the present application further with reference to the drawings.

Example 1: disruption effects of E.coli genomic DNA under disruption buffer containing calcium, magnesium, and manganese ions the present example uses E.coli genomic DNA of different initial amounts and evaluates the hairpin structure of the library under disruption buffer containing calcium, magnesium, and manganese ions.

1) E.coli genome fragmentation, end repair and addition of A.

The following libraries 1 and 2 are libraries constructed by breaking buffers containing calcium ions, magnesium ions, and manganese ions at the same time.

Disruption enzyme a (combinatorial enzyme) composition: DNase I (deoxyribonuclease I, a restriction enzyme) was used in an amount of 0.001U/. Mu.L; the Klenow DNA polymerase (E.coli DNA polymerase) was used in an amount of 0.2U/. Mu.L; taq DNA polymerase was used in an amount of 0.5U/. Mu.L.

Break bufferA (containing breaking reactants) composition: 300mM TrisHCl pH 8.5, 400mM NaCl, 7mM MgCl ₂、5mM CaCl₂、0.5mM MnCl₂, 0.5mM dNTPs (deoxynucleotides).

The E.coli genome was fragmented by adding reaction buffers according to the following table.

TABLE 1 disruption of the initial amount of the different E.coli genomes

The reaction conditions are as follows: all libraries were kept at 37℃for 10min and 65℃for 30min to give the dA-Tailing product, which was then stored at 4 ℃.

The total volume of the reaction system for each library disruption in Table 1 was 50. Mu.l, and the concentrations of each of the components in libraries 1 to 2 were: DNase I concentration is 0.0001U/. Mu.L, klenow DNA polymerase concentration is 0.02U/. Mu.L, taq DNA polymerase dosage is 0.05U/. Mu.L, 30mM TrisHCl,40mM NaCl,0.7mM MgCl ₂,0.5mM CaCl₂,0.05mM MnCl₂, 0.05mM dNTPs.

2) After the breaking, the terminal repairing and the adding A are completed, the connection of the joints can be performed. Library 1 and library 2 were joined into a synthetic reaction system according to the configuration of Table 2.

The library was subjected to adaptor ligation, and the adaptor primer was a double-ended Index adaptor (illuminea platform) synthesized from the root. The components in Table 2 are described below.

DA-Tailing product: DNA after addition of A.

Adapter: and (3) a joint.

5X Fast Ligation Buffer (ligation reaction solution): 200mM TrisHCl, pH 7.5, 20mM MgCl ₂, 10mM ATP,10%PEG8000 (v/v).

T4 DNA LIGASE (600U/. Mu.l) was derived from TIANSeq T DNA ligase (fast) (NG 201).

The configuration of the joint connection system was performed according to the following table 2.

TABLE 2 ligation reaction System

The reaction conditions are as follows: kept at 20℃for 15min and stored at 4 ℃.

After the ligation reaction was completed, library purification was performed after ligation: adding No. 1-2 library into a PCR tube, adding 60 mu L of rhizoma TIANSEQ DNA fragment sorting and purifying magnetic beads (NG 316), mixing uniformly, standing at room temperature, and incubating for 5min; placing the PCR tube on a magnetic rack, and sucking the supernatant after the solution is clarified; 200. Mu.L of freshly prepared 80% ethanol was added and left to stand for 30s, and the supernatant was aspirated; adding 200 mu L of freshly prepared 80% ethanol, standing for 30s, and sucking the ethanol; standing at room temperature for 3min, air drying ethanol, and adding 22 μl of nuclease-free water suspension magnetic beads; standing at room temperature for 5min, placing the PCR tube on a magnetic rack, and sucking out 20 mu L of supernatant after the solution is clarified, wherein the purified joint connection product is contained.

3) Library amplification

Library amplification enzyme used for library nos. 1-2 was the Tiangen TIANSEQ NGS library enrichment module (NG 304). The Tiangen TIANSEQ NGS library enrichment module (NG 304) included 2X HIFI PCRMASTER Mix and P5/P7 Primers Mix. The reaction system for library amplification is shown in Table 3.

TABLE 3 library amplification reaction System

The reaction procedure was:

Library 1:98℃2min,12cycles (98℃20s,60℃30s,72℃30 s), 72℃1min, and storage at 4 ℃.

Library 2:98℃2min,6cycles (98℃20s,60℃30s,72℃30 s), 72℃1min, and storage at 4 ℃.

The initial amounts of E.coli genomic DNA were different for library 1 and library 2, so the amplification reaction procedure was also adjusted accordingly.

Library purification was performed after the amplification reaction was completed: adding 50 mu L of a TIANSEQ DNA fragment of a No.1 library and a No. 2 library in different PCR tubes respectively, sorting and purifying magnetic beads (NG 316), mixing uniformly, standing at room temperature, and incubating for 5min; placing the PCR tube on a magnetic rack, and sucking the supernatant after the solution is clarified; 200. Mu.L of freshly prepared 80% ethanol was added and left to stand for 30s, and the supernatant was aspirated; 200. Mu.L of freshly prepared 80% ethanol was added and left to stand for 30s, and the ethanol was blotted dry. Standing at room temperature for 3min, air drying ethanol, and adding 22 μl of nuclease-free water suspension magnetic beads. Standing at room temperature for 5min, placing the PCR tube on a magnetic rack, sucking out 20 mu L of supernatant to obtain purified library after the solution is clarified, and finally quantifying and quality testing the library.

Library concentrations are shown in Table 4, with 2100 peak plots for library 1 and library 2 shown in FIG. 1. The ordinate of fig. 1 is FU (fluorescence unit), and the abscissa is s (seconds, retention time).

TABLE 4 library yield

4) MAPPED READS (base sequences aligned to the reference genome) the number of hairpin structures per M reads (per million base sequences) is shown in Table 5.

TABLE 5 number of hairpin structures per M reads in mapped reads

MAPPED READS (%): the ratio of reads aligned to the reference genome (including single-ended alignment and double-ended alignment).

Uniq mapped (%): the reads to unique positions of the reference genome are aligned.

AVERAGE DEPTH (x): average sequencing depth, total number of bases aligned to the reference genome divided by genome size.

Genome Coverage (%): genome coverage.

Uniformity (%): the uniformity of distribution of the data obtained by sequencing across the genome or region of interest. The higher the uniformity, the better, indicating that each site was uniformly measured to a substantially uniform depth.

Duplication (%): redundancy rate.

Chimera (%): percentage of chimeras.

Hairpin structures/M: number of hairpin structures per million reads.

As can be seen from the reaction conditions of example 1 and Table 5, the amount of hairpin structures in each million reads of the library reaches 12-18 ten thousand under the action of breaking buffer containing calcium ions, magnesium ions and manganese ions, and the generation amount of hairpin structures is large and the ratio of hairpin structures is too high.

Example 2: disruption effect of E.coli genomic DNA under the action of disruption buffer containing only calcium ion and magnesium ion this example uses E.coli genomic DNA of different initial amounts to evaluate the hairpin structure of the library under the action of disruption buffer containing calcium ion and magnesium ion. Example 2 eliminates the use of manganese ions compared to example 1.

1) E.coli genome fragmentation, end repair and addition of A.

Library 3 and library 4 are libraries constructed by interrupting buffer containing only calcium ions and magnesium ions according to the present application.

Disruption enzyme a (combinatorial enzyme) composition: DNase I was used in an amount of 0.001U/. Mu.L; klenow DNA polymerase was used in an amount of 0.2U/. Mu.L; taq DNA polymerase was used in an amount of 0.5U/. Mu.L. The disruption enzyme A of example 2 has the same composition as the disruption enzyme A of example 1.

Break buffer B (containing breaking reactant) composition: 300mM TrisHCl pH 8.5, 400mM NaCl, 7mM MgCl ₂、5mM CaCl₂, 0.5mM dNTPs. The break buffer B of example 2 is different from the break bufferA of example 1, and example 2 removes MnCl ₂.

The E.coli genomic DNA was fragmented by adding a reaction buffer according to the following table.

TABLE 6 disruption of the initial amount of E.coli genomes

The reaction conditions are as follows: all libraries were kept at 37℃for 10min and 65℃for 30min, and immediately after the reaction at 4℃to give the dA-Tailing product.

The proportions and reaction conditions of Table 6 are substantially the same as those of Table 1 and the reaction conditions thereof in example 1, except that Table 6 replaces the break bufferA of Table 1 with a break buffer B.

The total volume of the reaction system for each library disruption in Table 6 was 50. Mu.l, and the concentrations of each of the components in libraries 3 to 4 were: DNase I concentration is 0.0001U/. Mu.L, klenow DNA polymerase concentration is 0.02U/. Mu.L, taq DNA polymerase dosage is 0.05U/. Mu.L, 30mM TrisHCl,40mM NaCl,0.7mM MgCl ₂,0.5mM CaCl₂, 0.05mM dNTPs.

2) After the breaking, the terminal repairing and the adding A are completed, the connection of the joints can be performed. The libraries No. 3 to No. 4 were connected to a synthesis reaction system according to the configuration of Table 7.

The library Nos. 3 to 4 were ligated by adaptors, and the adaptor primer was a double-ended Index adaptor (illuminea platform) synthesized from Tiangen.

5× Fast Ligation Buffer composition: 200mM TrisHCl, pH 7.5, 20mM MgCl ₂, 10mM ATP,10%PEG8000 (v/v).

The configuration of the joint connection system was performed according to the following table 7.

TABLE 7 ligation reaction System

The reaction conditions are as follows: 20 ℃ for 15min, and stored at 4 ℃.

After the ligation reaction was completed, library purification was performed after ligation: adding 60 mu L of rhizoma at TIANSEQ DNA fragments of sorting and purifying magnetic beads (NG 316) into the No. 3-4 libraries in the two groups of PCR tubes respectively, mixing uniformly, standing at room temperature, and incubating for 5min; placing the PCR tube on a magnetic rack, and sucking the supernatant after the solution is clarified; 200. Mu.L of freshly prepared 80% ethanol was added and left to stand for 30s, and the supernatant was aspirated; adding 200 mu L of freshly prepared 80% ethanol, standing for 30s, and sucking the ethanol; standing at room temperature for 3min, air drying ethanol, and adding 22 μl of nuclease-free water suspension magnetic beads; standing at room temperature for 5min, placing the PCR tube on a magnetic rack, sucking out 20 mu L of supernatant after the solution is clarified to obtain purified joint connection product, and carrying out library amplification.

3) Library amplification

Library amplification enzymes used for libraries No. 3-4 were the Tiangen TIANSEQ NGS library enrichment module (NG 304), tiangen TIANSEQ NGS library enrichment module (NG 304) comprised 2X HIFI PCRMASTER Mix and P5/P7 Primers Mix. The reaction system for library amplification is shown in Table 8.

Table 8 library amplification reaction System

The reaction procedure was:

library 3:98℃2min,12cycles (98℃20s,60℃30s,72℃30 s), 72℃1min, and storage at 4 ℃.

Library 4:98℃2min,6cycles (98℃20s,60℃30s,72℃30 s), 72℃1min, and storage at 4 ℃.

Since the initial amounts of E.coli genomic DNA were different in library 3 and library 4, the above amplification reaction procedure was also adjusted accordingly.

Library purification was performed after the completion of the above PCR: adding 50 mu L of rhizoma at TIANSEQ DNA fragments to separate and purify magnetic beads (NG 316) into the No. 3-4 libraries placed in the two groups of PCR tubes respectively, mixing uniformly, standing at room temperature, and incubating for 5min; placing the PCR tube on a magnetic rack, and sucking the supernatant after the solution is clarified; 200 mu L of freshly prepared 80% ethanol is respectively added and kept stand for 30s, and the supernatant is sucked; 200 mu L of freshly prepared 80% ethanol is respectively added and kept stand for 30s, and the ethanol is sucked dry; standing at room temperature for 3min, air drying ethanol, adding 22 μl of non-nuclease water suspension magnetic beads respectively, standing at room temperature for 5min, placing PCR tube on a magnetic rack, sucking out 20 μl of supernatant after solution is clarified to obtain purified library 3 and library 4, and performing the following library quantification and quality inspection.

The concentrations of libraries 3 and 4 are shown in Table 9, and 2100 peak plots for library 3 and library 4 are shown in FIG. 2. The ordinate of fig. 2 is FU (fluorescence unit), and the abscissa is s (seconds, retention time).

TABLE 9 library yield

4) The number of hairpin structures per M reads in MAPPED READS is shown in Table 10.

TABLE 10 number of hairpin structures per M reads in mapped reads

As can be seen from the reaction conditions of example 2 and Table 10, the number of hairpin structures per million reads in the library under the action of the break buffer containing calcium and magnesium ions was 689-2058 in the E.coli genomic DNA. The hairpin structure of this example 2 is less occupied than example 1.

Example 3: disruption of E.coli genomic DNA with the action of T4GP32 (T4 single-stranded DNA binding protein) containing different concentrations

This example uses the same initial amount of E.coli genomic DNA to evaluate the hairpin formation of the library under the action of a disruption enzyme containing different concentrations of T4GP 32.

1) E.coli genome fragmentation, end repair and addition of A.

Library 5, library 6, library 7 are library constructs of the application under the action of disruption enzymes containing different concentrations of T4GP 32.

Disruption enzyme a (combinatorial enzyme) composition: DNase I was used in an amount of 0.001U/. Mu.L; klenow DNA polymerase was used in an amount of 0.2U/. Mu.L; taq DNA polymerase was used in an amount of 0.5U/. Mu.L. The composition of the interrupting enzyme A is the same as that of the interrupting enzymes A in the example 1 and the example 2.

Disruption enzyme A1 (combinatorial enzyme) composition: DNase I was used in an amount of 0.001U/. Mu.L; klenow DNA polymerase was used in an amount of 0.2U/. Mu.L; the dosage of Taq DNA polymerase is 0.5U/. Mu.L; the amount of T4GP32 was 0.6. Mu.g/. Mu.L.

Disruption enzyme A2 (combinatorial enzyme) composition: DNase I was used in an amount of 0.001U/. Mu.L; klenow DNA polymerase was used in an amount of 0.2U/. Mu.L; the dosage of Taq DNA polymerase is 0.5U/. Mu.L; the amount of T4GP32 was 2. Mu.g/. Mu.L.

Break bufferB (containing breaking reactants) composition: 300mM TrisHCl pH 8.5, 400mM NaCl, 7mM MgCl ₂、5mM CaCl₂, 0.5mM dNTPs. The interruption bufferB of this example 3 is identical to the interruption bufferB of example 2.

TABLE 11 disruption of 20ng E.coli genome by different disruption enzymes

The reaction conditions of libraries 5 to 7 are: after reaction, dA-Tailing product is obtained after reaction at 37 ℃ for 10min and 65 ℃ for 30min, and the product is stored at4 ℃. The reaction conditions were the same as in example 1 and example 2, but the amount of E.coli genomic DNA was different.

Each of the libraries shown in Table 11 above was broken down into a reaction system in a total volume of 50. Mu.l. The concentrations of the components of the same parts of libraries 5 to 7 were: DNase I concentration is 0.0001U/. Mu.L, klenow DNA polymerase concentration is 0.02U/. Mu.L, taq DNA polymerase dosage is 0.05U/. Mu.L, 30mM TrisHCl,40mM NaCl,0.7mM MgCl ₂,0.5mM CaCl₂, 0.05mM dNTPs. The concentrations of the components of the different parts of libraries 5 to 7 were: library 5 did not contain T4GP32, and library 6 had a T4GP32 concentration of 0.06 μg/μL and library 7 had a T4GP32 concentration of 0.2 μg/μL.

The following ligation and amplification reaction conditions were the same as those of example 1 and example 2.

2) After the breaking, the terminal repairing and the adding A are completed, the connection of the joints can be performed. Library nos. 5 to 7 were connected to a synthesis reaction system according to the configuration of table 12.

The libraries No. 5-7 were ligated, and the adaptor primer was a double-ended Index adaptor (illuminea platform) synthesized from the root.

The configuration of the joint connection system was performed according to the following table 12.

Table 12 connection reaction System

The reaction conditions for the three libraries were: the reaction mixture was kept at 20℃for 15min and then stored at 4 ℃.

After the ligation reaction was completed, the library was purified after ligation. Adding 60 mu L of rhizoma et radix Barbatae TIANSEQ DNA fragment, separating and purifying magnetic beads (NG 316) into the No. 5-7 libraries in the three groups of PCR tubes respectively, mixing uniformly, standing at room temperature, and incubating for 5min; placing the PCR tube on a magnetic rack, and sucking the supernatant after the solution is clarified; 200. Mu.L of freshly prepared 80% ethanol was added and left to stand for 30s, and the supernatant was aspirated; adding 200 mu L of freshly prepared 80% ethanol, standing for 30s, and sucking the ethanol; standing at room temperature for 3min, air drying ethanol, adding 22 μl of nuclease-free water suspension magnetic beads, and standing at room temperature for 5min; the PCR tube was placed on a magnetic rack, and after the solution was clarified, 20. Mu.L of the supernatant containing the purified adaptor-ligated product was aspirated for the following library amplification.

3) Library amplification

Library amplification enzyme used for libraries No. 5-7 was the Tiangen TIANSEQ NGS library enrichment module (NG 304). The Tiangen TIANSEQ NGS library enrichment module (NG 304) included 2X HIFI PCRMASTER Mix and P5/P7 Primers Mix. The reaction system for library amplification is shown in Table 13.

TABLE 13 library amplification reaction System

The reaction procedure was: 98℃2min,12cycles (98℃20s,60℃30s,72℃30 s), 72℃1min, and storage at 4 ℃.

Library purification was performed after the completion of the PCR. Adding 50 mu L of rhizoma et radix Barbatae TIANSEQ DNA fragment to separate and purify magnetic beads (NG 316) into the No. 5-7 libraries in the three groups of PCR tubes respectively, mixing uniformly, standing at room temperature, and incubating for 5min; placing the PCR tube on a magnetic rack, and sucking the supernatant after the solution is clarified; 200. Mu.L of freshly prepared 80% ethanol was added and left to stand for 30s, and the supernatant was aspirated; adding 200 mu L of freshly prepared 80% ethanol, standing for 30s, and sucking the ethanol; standing at room temperature for 3min, air drying ethanol, adding 22 μl of nuclease-free water suspension magnetic beads, standing at room temperature for 5min, placing PCR tube on a magnetic rack, and sucking out 20 μl of supernatant after the solution is clear for library quantification and quality inspection.

The concentrations of libraries 5-7 are shown in Table 14, and the 2100 peak plots for libraries 5-7 are shown in FIG. 3. The ordinate of fig. 3 is FU (fluorescence unit), and the abscissa is s (seconds, retention time).

TABLE 14 library yield

4) The number of hairpin structures per M reads in MAPPED READS is shown in Table 15.

TABLE 15 number of hairpin structures per M reads in mapped reads

As can be seen from the reaction conditions of example 3 and Table 15, the same initial amount of E.coli genomic DNA, under the action of a disruption buffer containing the same concentration of calcium ions and magnesium ions, and under the action of a disruption enzyme containing different concentrations of T4GP32, the number of hairpin structures per million reads of the library is 278-872, the amount of T4GP32 is 0-2. Mu.g/. Mu.L, and the larger the amount of T4GP32 in the disruption enzyme, the smaller the number of hairpin structures of the library, such as Website 7.

Thus, the components and their concentrations in the present fragmenting enzyme reaction system are selected as: DNase I concentration is 0.00005-0.0005U/. Mu.L, klenow DNA polymerase concentration is 0.01-0.03U/. Mu.L, taq DNA polymerase concentration is 0.03-0.07U/. Mu.L, trisHCl concentration is 10-50 mM, naCl concentration is 20-60 mM, mgCl ₂ concentration is 0.5-0.9 mM, caCl ₂ concentration is 0.3-0.7 mM, dNTPs concentration is 0.03-0.07 mM, and the kit does not contain Mn ions.

Further preferably, DNase I is 0.0001U/. Mu.L, klenow DNA polymerase is 0.02U/. Mu.L, taq DNA polymerase is used in an amount of 0.05U/. Mu.L, and T4GP32 is 0.06-0.2. Mu.g/. Mu.L, 30mM TrisHCl,40mM NaCl,0.7mM MgCl ₂,0.5mM CaCl₂, 0.05mM dNTPs.

The above embodiments are merely examples of the present application, and the protection scope of the present application is not limited to the above embodiments, and it should be obvious to those skilled in the art that several modifications and variations are possible without departing from the inventive concept.

Claims

1. A fragmenting enzyme reaction system for reducing the number of hairpin structures formed by DNA disruption, said fragmenting enzyme reaction system comprising: combining enzymes and breaking reactants; the combination enzyme comprises restriction enzyme and polymerase; the breaking reactants comprise Mg ²⁺ and Ca ²⁺, and are free of manganese ions; in the fragmenting enzyme reaction system, the concentration of Mg ²⁺ is 0.5-0.9 mM, and the concentration of Ca ²⁺ is 0.3-0.7 mM.

2. The system of claim 1, wherein the combinatorial enzyme further comprises a T4 single-stranded DNA binding protein.

3. The system of claim 2, wherein the concentration of the T4 single-stranded DNA binding protein in the system is 0.06-0.2 μg/μl.

4. A fragmenting enzyme reaction system for reducing the number of hairpin structures formed by DNA fragmentation according to any one of claims 1 to3, wherein the fragmentation reactants comprise TrisHCl, naCl, mgCl ₂、CaCl₂ and dNTPs; the concentration of each component of the breaking reactant in the fragmenting enzyme reaction system is as follows: the concentration of TrisHCl is 10-50 mM, the concentration of NaCl is 20-60 mM, the concentration of MgCl ₂ is 0.5-0.9 mM, the concentration of CaCl ₂ is 0.3-0.7 mM, and the concentration of dNTPs is 0.03-0.07 mM.

5. The system of claim 1, wherein the restriction enzyme is one or both of DNase I and dsDNase.

6. The system of claim 5, wherein the polymerase is selected from one or more of Klenow DNA polymerase, taq DNA polymerase, and BST DNA polymerase.

7. The system of claim 6, wherein the restriction enzyme is DNase I; the polymerase is a combination of Klenow DNA polymerase and Taq DNA polymerase; in the reaction system of the fragmenting enzyme: DNase I concentration is 0.00005-0.0005U/. Mu.L, klenow DNA polymerase concentration is 0.01-0.03U/. Mu.L, taq DNA polymerase concentration is 0.03-0.07U/. Mu.L.

8. A DNA library construction method, characterized in that a DNA library is constructed by using the fragmenting enzyme reaction system according to any one of claims 1 to 7; the DNA library construction method comprises the following steps:

S3, purifying the DNA ligation product;

9. The method of constructing a DNA library according to claim 8, wherein the reaction conditions in step S1 are: mixing the DNA sample with the reaction system of the fragmenting enzyme, keeping the temperature at 36-38 ℃ for 5-15 min, and then keeping the temperature at 60-70 ℃ for 20-40 min, so that the DNA is continuously fragmented, the tail end is repaired, and the 3' -end is added with A, and a DNA added A product is obtained.