CN114196661B - Recombinant topoisomerase and application thereof in construction of sequencing library - Google Patents
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Abstract
The invention discloses a recombinant topoisomerase and application thereof in constructing a sequencing library. The invention firstly discloses recombinant topoisomerase with an amino acid sequence shown as SEQ ID NO. 1. The invention further discloses application of the recombinant topoisomerase in construction of a sequencing library. The invention creatively applies the topoisomerase to the DNA library system of the enzyme digestion method, so that the efficiency of fragmenting, terminal repairing and 3' end-A adding treatment of the DNA sample is higher and balance is easier to achieve; in addition, the problems that the end repair is incomplete due to the rapid annealing of single strands in the enzyme digestion reaction in the construction process of the sequencing library, the GC distribution of a DNA high GC region in the sequencing is unbalanced and the conversion efficiency of a library is low when the initial sample amount is low are solved by the action of DNA melting and cutting with the participation of recombinant topoisomerase, the yield and the quality of the sequencing library are improved, and the method has wide applicability and convenient operability.
Description
Technical Field
The present invention relates to the field of biotechnology. More particularly, to a recombinant topoisomerase and its use in constructing sequencing libraries.
Background
With the rapid development of molecular diagnosis technology, the application field is more and more, and particularly with the development of Metagnomic Next-Generation Sequencing (mNGS), the demand of rapid DNA library establishment is more and more urgent. In general, NGS pooling includes (1) nucleic acid (DNA or RNA) fragmentation; (2) screening for fragmentation size; (3) adding a linker to the fragmented nucleic acid; (4) Sequencing (Behjati S, tarpey PS.What is next generation sequencing. As the first step of NGS library construction, which is also the most critical one step of nucleic acid fragmentation, two methods of mechanical ultrasonic disruption and cleavage are currently mainly used (Nelly Sapojnikova, nino Asiania, tamar Kartvelishvili, lali Asishvili, vitaly Zinkevich, irina Bogdalina, julian Mitchell, abdulmohsen Al-Humam, A comparison of DNA fragmentation methods-Applications for the biochip technology, journal of Biotechnology, volume 256,2017, pages 1-5.). Because the mechanical ultrasonic breaking instrument is expensive, large in occupied area, low in flux, complicated in operation and long in time, the required sample amount is large (the trace sample fragmentation loss is large), the rapid processing efficiency of DNA library construction on multiple samples is severely limited, and the popularization of the DNA library construction in the medical field is hindered. The use of DNA fragmenting enzyme (i.e. enzyme digestion) effectively solves the problem of mechanical ultrasonic disruption: the wide template input amount range (100 pg-1 mug) and controllable DNA fragmentation in the PCR tube meet the requirements of DNA library establishment on high flux, simple operation and low DNA input amount.
The current DNA library construction by the enzyme digestion method is based on the following principle: 1>Randomly nicking the DNA using a nicking enzyme, while using T7 endonucleolytic I to identify and cleave the nicking point; 2>Use of DNase I or dsDNase at very low levels of Mg 2+ And Mn of 2+ Fragmenting dsDNA under the action of a primer; 3>Fragmenting dsDNA using a plurality of restriction enzyme combinations; 4>The use of VVN or DNase I or dsDNase at very low amounts exploits the strand displacement capabilities of the polymerase for fragmentation. However, in either method, there is a preference for fragmentation (limited by preference of nuclease) and a GC distribution unevenness in the high GC region (single strand formed after cleavage of the high GC region is easily annealed to double strand itself); for SV and SNP determination and identification of the library, a certain influence was caused (Chen Y-C, liu T, yu C-H, chiang T-Y, hwang C-C (2013) Effects of GC Bias in Next-Generation-Sequencing Data on De Novo Genome analysis.PLoS ONE 8 (4): e 62856.).
Topoisomerase catalyzes a coupling reaction of breaking and binding DNA strands, and can cut off DNA of one strand or two strands (cut at a place to be knotted or knotted) and reset a ligation function (Natassja G Bush, katherane Evans-Roberts, anthony Maxwell. DNA Topoisomerases. EcoSal plus.2015;6 (2) doi: 10.1128/ecosystem. ESP-0010-2014.) so that circular DNA formed by itself can be effectively cut, and the defect of the technology for constructing an enzymatic DNA library is a powerful improvement. However, topoisomerase can reset the ligation in addition to being able to cleave, which is not required.
Thus, there is a need to provide a novel topoisomerase for use in the construction of sequencing libraries.
Disclosure of Invention
The first object of the present invention is to provide a recombinant topoisomerase, which is combined with the existing enzyme digestion reaction solution for constructing a sequencing library, so as to effectively improve the yield and sequencing data quality of the library constructed by the enzyme digestion sequencing library.
The second object of the present invention is to provide a cleavage reaction solution comprising the recombinant topoisomerase.
The third object of the present invention is to provide a kit comprising the recombinant topoisomerase or the cleavage reaction solution.
The fourth object of the invention is to provide the use of the recombinant topoisomerase, enzyme cleavage reaction solution or kit described above in the construction of sequencing libraries.
A fifth object of the present invention is to provide a method of constructing a sequencing library.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a recombinant topoisomerase having an amino acid sequence as shown in SEQ ID NO. 1.
The recombinant topoisomerase disclosed by the invention is obtained by carrying out mutation transformation on the E.coli type I topoisomerase (the amino acid sequence is shown as SEQ ID NO. 6), so that the reset connection function of the enzyme is greatly reduced/deleted, the DNA cutting function of the enzyme is acted on the construction of a sequencing library, the DNA fragmentation, the terminal modification and the 3' A tail addition reaction are involved, the heterogeneity of GC distribution in the conventional library construction is greatly improved, and the preference of the GC distribution in the sequencing analysis is more consistent in a high GC region.
In a second aspect, the invention provides an enzyme digestion reaction solution comprising a mixed solution of fragmenting enzymes, wherein the mixed solution of fragmenting enzymes comprises the recombinant topoisomerase.
Further, the mixed solution of the fragmenting enzyme further comprises endonuclease and DNA polymerase.
The recombinant topoisomerase in the enzyme digestion reaction liquid can be combined with different endonucleases (used for cutting or nicking DNA double chains) and comprises any one or two of double-chain DNA nuclease and ATP dependent nuclease; the recombinant topoisomerase does not affect the digestion effect of endonuclease.
The recombinant topoisomerase in the enzyme digestion reaction liquid can be combined with different DNA polymerases (used for carrying out end repair on the cut nucleic acid or carrying out A tail addition reaction on the 3' end of the cut nucleic acid), and the recombinant topoisomerase comprises any one or two of low-temperature DNA polymerase and heat-resistant DNA polymerase; the low-temperature DNA polymerase comprises any one or the combination of two of Phi29DNA polymerase and T4 DNA polymerase large fragment Klenow; the heat-resistant DNA polymerase comprises any one or the combination of two of Bst II DNA polymerase and Taq DNA polymerase large fragment Klenow; the recombinant topoisomerase does not affect the polymerization effect of the DNA polymerase.
Further, the mixed solution of the fragmenting enzymes further comprises auxiliary proteins, wherein the auxiliary proteins are used for regulating the cutting rate of endonuclease and the end repair rate of DNA polymerase.
The recombinant topoisomerase in the enzyme digestion reaction liquid can be combined with a plurality of auxiliary proteins, wherein the auxiliary proteins comprise any one or combination of two of recombinant albumin (BSA substitute) and single-chain binding protein (SSB); the recombinant topoisomerase does not affect the regulatory function of these accessory proteins.
In a specific embodiment of the invention, the cocktail consists of the recombinant topoisomerase described above (amino acid sequence shown in SEQ ID NO. 1), ATP dependent nuclease (amino acid sequence shown in SEQ ID NO. 2), phi29DNA polymerase (TransGen, LP 101), bst II DNA polymerase (TransGen, LP 301), SSB (amino acid sequence shown in SEQ ID NO. 4), recombinant albumin (amino acid sequence shown in SEQ ID NO. 3), dsDNase (TransGen, LD 101), tris-HCl, KCl, tween 20, DTT and glycerol.
In a preferred embodiment of the invention, the final concentration of the recombinant topoisomerase in the mixture of fragmenting enzymes is 4 ng/. Mu.l to 15 ng/. Mu.l; the final concentration of the ATP dependent nuclease in the fragmenting enzyme mixture is 0.4 ng/. Mu.l to 0.8 ng/. Mu.l; the final concentration of the Phi29DNA polymerase in the fragmenting enzyme mixed solution is 15 ng/mu l-25 ng/mu l; the final concentration of the Bst II DNA polymerase in the fragmenting enzyme mixed solution is 7.5 ng/. Mu.l to 15 ng/. Mu.l; the final concentration of the SSB in the fragmenting enzyme mixture is 120 ng/. Mu.l to 300 ng/. Mu.l; the final concentration of the recombinant albumin in the fragmenting enzyme mixed solution is 300 ng/mu l-700 ng/mu l; the final concentration of the dsDNase in the mixed solution of the fragmenting enzyme is 0.6 ng/. Mu.l to 1.0 ng/. Mu.l; the final concentration of the Tris-HCl in the fragmenting enzyme mixed solution is 40mM-60mM; the final concentration of KCl in the fragmenting enzyme mixed solution is 15mM-40mM; the final concentration of the Tween 20 in the fragmenting enzyme mixed solution is 0.25% -0.75% (V/V); the final concentration of the DTT in the fragmenting enzyme mixed solution is 0.5mM-2mM; the final concentration of the glycerol in the fragmenting enzyme mixture is 40% -60% (V/V).
In a more preferred embodiment of the invention, the final concentration of the recombinant topoisomerase in the mixture of fragmenting enzymes is 11.0 ng/. Mu.l; the final concentration of the ATP dependent nuclease in the fragmenting enzyme mix is 0.6 ng/. Mu.l; the final concentration of the Phi29DNA polymerase in the fragmenting enzyme mixed solution is 20 ng/. Mu.l; the final concentration of the Bst II DNA polymerase in the fragmenting enzyme mixed solution is 10 ng/. Mu.l; the final concentration of the SSB in the fragmenting enzyme mixture is 200 ng/. Mu.l; the final concentration of the recombinant albumin in the fragmenting enzyme mixed solution is 500 ng/. Mu.l; the final concentration of the dsDNase in the mixed solution of the fragmenting enzymes is 0.8 ng/. Mu.l; the final concentration of the Tris-HCl in the fragmenting enzyme mixed solution is 50mM; the final concentration of KCl in the fragmenting enzyme mixture is 25mM; the final concentration of Tween 20 in the fragmenting enzyme mixture is 0.5% (V/V); the final concentration of the DTT in the fragmenting enzyme mixture is 1mM; the final concentration of the glycerol in the fragmenting enzyme mixture was 50% (V/V).
The recombinant topoisomerase can be concentrated in a reaction system with the existing endonuclease for cutting or nicking DNA double chains and DNA polymerase for carrying out end repair on cut nucleic acid or carrying out A tail adding reaction on the 3' end of the cut nucleic acid, and the self-hybridized double-chain DNA formed by exposed single-chain DNA in a cut nucleic acid sample is melted, so that the preference of a high GC part in sequencing of a constructed sequencing library is effectively avoided.
Further, the enzyme digestion reaction liquid also comprises an enzyme digestion reaction buffer solution. The digestion reaction buffer of the present invention may be one commonly used in the art. The enzyme digestion reaction buffer solution comprises metal cations, a substrate and a buffer medium. The metal cations include Mg 2+ 、K + And NH 3 + Any one or more combinations of these, e.g. MgCl 2 KAc; the substrate comprises any one or more of dNTPs, dATP and ATP, such as dNTPs, dATP and ATP; the buffer medium comprises any one or more of 2-morpholinoethanesulfonic acid (MES), acetic acid (Ac) and Tris (Tris), such as Tris-acetic acid; the cleavage reaction buffer can perform a melting action without causing inhibition or inhibition to the above-mentioned mixed solution of the fragmenting enzymes.
In a specific embodiment of the present invention, the cleavage reaction buffer consists of Tris-acetate, mgCl 2 KAc, dNPTs, dATP and ATP.
In a preferred embodiment of the present invention, the final concentration of said Tris-acetate in said cleavage reaction buffer is 200mM-400mM; the MgCl 2 The final concentration in the cleavage reaction buffer is 75mM-125mM; the final concentration of KAc in the digestion reaction buffer is 100mM-300mM; the final concentration of the dNTPs in the digestion reaction buffer is 1.5mM-3mM; the final concentration of the dATP in the digestion reaction buffer is 3mM-7mM; the final concentration of ATP in the cleavage reaction buffer is 20mM-35mM.
More preferably in the present inventionIn an embodiment of (2), the final concentration of Tris-acetate in the cleavage reaction buffer is 300mM; the MgCl 2 The final concentration in the cleavage reaction buffer is 100mM; the final concentration of KAc in the cleavage reaction buffer is 200mM; the final concentration of the dNTPs in the digestion reaction buffer is 2mM; the final concentration of dATP in the cleavage reaction buffer is 5mM; the final concentration of ATP in the cleavage reaction buffer is 25mM.
In a specific embodiment of the invention, the pH of the cleavage reaction buffer is between 7.5 and 8.5. In a preferred embodiment of the invention, the pH of the cleavage reaction buffer is 8.
The recombinant topoisomerase has strong adaptability to the combination of metal cation types, concentration and types and concentration of buffer media in an enzyme digestion reaction buffer solution within a certain range, has no influence on the activities of endonuclease and DNA polymerase, and has a melting function so that the palindromic structural region of a special high GC is easier to detect, thereby being more beneficial to base balance and avoiding GC separation.
In a third aspect, the invention provides a kit comprising the recombinant topoisomerase described above or the cleavage reaction solution described above.
Further, the kit further comprises a joint connection reaction liquid, wherein the joint connection reaction liquid comprises a joint ligase mixed liquid and a joint connection reaction buffer liquid.
The mixed liquid of the joint ligase in the kit can be the mixed liquid of the joint ligase commonly used in the prior art. In the present invention, the adaptor ligase mixture comprises a polynucleotide kinase and a ligase; the polynucleotide kinase may be a T4 polynucleotide kinase (T4 PNK) that 5' phosphorylates the cleaved nucleic acid; the ligase may be T4 DNA ligase, ligating the adaptor.
In a specific embodiment of the invention, the adaptor ligase mixture consists of T4 DNA ligase, T4 PNK, tris-Ac, tween 20, DTT and glycerol.
In a preferred embodiment of the invention, the final concentration of said T4 DNA ligase in said adaptor ligase mixture is 600 ng/. Mu.l to 1. Mu.g/. Mu.l; the final concentration of the T4 PNK in the joint ligase mixed solution is 30 ng/. Mu.l-100 ng/. Mu.l; the final concentration of the Tris-Ac in the joint ligase mixed solution is 30mM-100mM; the final concentration of Tween 20 in the joint ligase mixed solution is 0.25% -0.7% (V/V); the final concentration of the DTT in the joint ligase mixed solution is 0.5mM-2mM; the final concentration of the glycerol in the adaptor ligase mixed solution is 40% -60% (V/V).
In a more preferred embodiment of the invention, the final concentration of said T4 DNA ligase in said adaptor ligase mixture is 800 ng/. Mu.l; the final concentration of the T4 PNK in the joint ligase mixed solution is 80 ng/. Mu.l; the final concentration of Tris-Ac in the adaptor ligase mixed solution is 50mM; the final concentration of Tween 20 in the adaptor ligase mixture is 0.5% (V/V); the final concentration of the DTT in the adaptor ligase mixture is 1mM; the final concentration of the glycerol in the adaptor ligase mixture was 50% (V/V).
The linker ligation reaction buffer in the present invention may be a linker ligation reaction buffer commonly used in the art; in a specific embodiment of the invention, the adaptor ligation reaction buffer is Adapter Ligation Buffer in TransGen#KP201TransNGS DNA Library Prep Kit for Illumina.
Further, the kit also comprises any one or more of a sequencing joint, a PCR amplification enrichment reagent and a purification reagent.
In a fourth aspect, the invention provides the use of the recombinant topoisomerase, cleavage reaction solution or kit described above in the construction of a sequencing library or in the preparation of reagents for the construction of a sequencing library.
In a fifth aspect, the present invention provides a method of constructing a sequencing library, the method comprising:
the target DNA is fragmented, the tail end is repaired and the 3' end A is added by utilizing the enzyme digestion reaction liquid containing the recombinant topoisomerase, the enzyme digestion reaction liquid or the enzyme digestion reaction liquid in the kit to obtain a fragmented product;
5' phosphorylation and linker ligation reaction are carried out on the fragmented product to obtain a phosphorylated and linker ligation product;
and carrying out PCR amplification enrichment on the phosphorylation and joint connection products to obtain a sequencing library.
Further, the reaction conditions of the fragmentation, the end repair and the 3' addition of the A tail are that the reaction is firstly incubated for 5 to 30 minutes at 32 ℃ or 37 ℃ and then incubated for 20 to 30 minutes at 65 ℃.
Further, the 5 'phosphorylation and linker ligation reaction is performed by the linker ligation reaction solution in the kit, and the reaction conditions of the 5' phosphorylation and linker ligation reaction are incubation at 25 ℃ for 15-20 minutes.
Further, the amount of the target DNA to be added is 100pg to 1. Mu.g.
Further, the construction method further comprises a step of purifying the obtained product after the adaptor-ligation reaction is performed.
Further, the construction method further comprises a step of purifying the obtained product after enrichment by PCR amplification.
The beneficial effects of the invention are as follows:
1) The invention creatively applies the topoisomerase to an enzyme digestion method DNA library building system, adds the recombinant topoisomerase into a fragmenting enzyme mixed solution in the process of constructing a sequencing library, and cooperates with metal cations and buffer media in an enzyme digestion reaction buffer solution to integrate DNA sample fragmentation, end repair and 3 'end A addition into one-step reaction, so that the cutting, end repair and 3' end A addition treatment efficiency of the enzyme digestion reaction solution on a nucleic acid sample are higher, and balance is easier to achieve; in addition, the problems that the end repair is incomplete due to the rapid annealing of single strands in the enzyme digestion reaction in the construction process of the sequencing library, the GC distribution of a DNA high GC region in the sequencing is unbalanced and the conversion efficiency of a library is low when the initial sample amount is low are solved by the action of DNA melting and cutting with the participation of recombinant topoisomerase, the yield and the quality of the sequencing library are improved, and the method has wide applicability and convenient operability.
2) According to the invention, 5' phosphorylation reacts with a joint connection system in the process of constructing a sequencing library, so that the joint which is failed in phosphorylation due to repeated freeze thawing is subjected to rephosphorization, the joint connection efficiency is improved, and the conversion rate of the library and the success rate of library construction are further improved on the basis of unchanged flow of enzyme digestion DNA library construction.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the drawings.
FIG. 1 is a 2100 peak profile of a sequencing library constructed in combination 1;
FIG. 2 is a 2100 peak profile of a sequencing library constructed in combination 2;
FIG. 3 is a 2100 peak profile of a sequencing library constructed in combination 3;
FIG. 4 is a 2100 peak profile of a sequencing library constructed in combination 5;
FIG. 5 is a 2100 peak profile of a sequencing library constructed in combination 6;
FIG. 6 is a 2100 peak profile of a sequencing library constructed in combination 7;
FIG. 7 is a graph showing GC content distribution and depth distribution after pooling using combinations 5, 6, and 7 for different DNA inputs; wherein, the DNA input amount of A was 100pg, the DNA input amount of B was 1ng, the DNA input amount of C was 10ng, the DNA input amount of D was 100ng, and the DNA input amount of E was 1. Mu.g.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. Like parts in the drawings are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Wherein, the sources of partial reagents in the examples are as follows:
ATP-dependent nucleases: the amino acid sequence is shown as SEQ ID NO. 2;
recombinant albumin (BSA substitute): the amino acid sequence is shown as SEQ ID NO. 3;
SSB (single chain binding protein): the amino acid sequence is shown as SEQ ID NO. 4;
taq DNA polymerase large fragment Klenow: the amino acid sequence is shown as SEQ ID NO. 5;
dsDNase：TransGen，LD101；
phi29DNA polymerase: transGen, LP101;
bst II DNA polymerase: transGen, LP301;
t4 DNA ligase: transGen, LL101;
T4 PNK：TransGen，LK101。
EXAMPLE 1 construction of recombinant topoisomerase
The recombinant topoisomerase is a novel topoisomerase modified according to escherichia coli type I topoisomerase (the amino acid sequence of the novel topoisomerase is shown as SEQ ID NO. 6), and specifically comprises the following components: the E.coli I topoisomerase is subjected to mutation transformation of enzyme evolution, the topoisomerase with the reset connection function is greatly reduced/deleted through screening, and the DNA cutting function is acted on the construction of a DNA library, so that the heterogeneity of the GC distribution of the traditional library construction is greatly improved.
Finally, the amino acid sequence of the obtained recombinant topoisomerase is shown as SEQ ID NO. 1.
Example 2 preparation of cleavage reaction solution
For better reaction topoisomerase function, the mixture of the fragmenting enzymes in the enzyme digestion reaction liquid is prepared into a mixture of fragmenting enzymes I without topoisomerase, a mixture of fragmenting enzymes II and III with recombinant topoisomerase as described in example 1, and a mixture of fragmenting enzymes IV with E.coli type I topoisomerase; the enzyme digestion reaction buffer solution is prepared finally.
1. Preparing a fragmenting enzyme mixed solution:
the components of the fragmenting enzyme mixture I and the final concentration of each component in the fragmenting enzyme mixture I: ATP dependent nuclease (0.6 ng/. Mu.l), dsDNase (0.8 ng/. Mu.l), recombinant albumin (BSA substitute, 400 ng/. Mu.l), phi29DNA polymerase (20 ng/. Mu.l), taq DNA polymerase large fragment Klenow (10 ng/. Mu.l), SSB (single-stranded binding protein, 200 ng/. Mu.l), tris-HCl (pH 8.0, 50 mM), KCl (25 mM), tween 20 (V/V, 0.5%), DTT (1 mM), glycerol (V/V, 50%).
Components of the mixture II of the fragmenting enzymes and final concentrations of the components in the mixture II of the fragmenting enzymes: the amino acid sequence described in example 1 is shown in SEQ ID NO.1 as recombinant topoisomerase (5.5 ng/. Mu.l), ATP dependent nuclease (0.6 ng/. Mu.l), dsDNase (0.8 ng/. Mu.l), recombinant albumin (BSA substitute, 500 ng/. Mu.l), phi29DNA polymerase (20 ng/. Mu.l), bst II DNA polymerase (10 ng/. Mu.l), SSB (single-stranded binding protein, 200 ng/. Mu.l), tris-HCl (pH8.0, 50 mM), KCl (25 mM), tween 20 (V/V, 0.5%), DTT (1 mM), glycerol (V/V, 50%).
Components of the mixture III of the fragmenting enzymes and final concentrations of the components in the mixture III of the fragmenting enzymes: the amino acid sequence described in example 1 is shown in SEQ ID NO.1 as recombinant topoisomerase (11.0 ng/. Mu.l), ATP dependent nuclease (0.6 ng/. Mu.l), dsDNase (0.8 ng/. Mu.l), recombinant albumin (BSA substitute, 400 ng/. Mu.l), phi29DNA polymerase (20 ng/. Mu.l), bst II DNA polymerase (10 ng/. Mu.l), SSB (single-stranded binding protein, 200 ng/. Mu.l), tris-HCl (pH8.0, 50 mM), KCl (25 mM), tween 20 (V/V, 0.5%), DTT (1 mM), glycerol (V/V, 50%).
Components of the mixture IV of the fragmenting enzymes and final concentrations of the components in the mixture IV of the fragmenting enzymes: the amino acid sequence described in example 1 is shown in SEQ ID NO.6 as Escherichia coli type I topoisomerase (11.0 ng/. Mu.l), ATP dependent nuclease (0.6 ng/. Mu.l), dsDNase (0.8 ng/. Mu.l), recombinant albumin (BSA substitute, 400 ng/. Mu.l), phi29DNA polymerase (20 ng/. Mu.l), bst II DNA polymerase (10 ng/. Mu.l), SSB (single-stranded binding protein, 200 ng/. Mu.l), tris-HCl (pH 8.0, 50 mM), KCl (25 mM), tween 20 (V/V, 0.5%), DTT (1 mM), glycerol (V/V, 50%).
2. Preparing enzyme digestion reaction buffer solution:
the final concentration of the components of the cleavage reaction buffer in the cleavage reaction buffer: tris-acetate (pH 8.0, 300 mM), mgCl 2 (100mM)，KAc(200mM)，dNPTs(2mM)，dATP(5mM)，ATP(25mM)。
3. Preparing enzyme cutting reaction liquid:
enzyme digestion reaction liquid 1: fragmenting enzyme mixed solution I+enzyme cutting reaction buffer solution;
enzyme digestion reaction liquid 2: fragmenting enzyme mixed solution II+enzyme cutting reaction buffer solution;
enzyme digestion reaction liquid 3: fragmenting enzyme mixed solution III+enzyme cutting reaction buffer solution.
Enzyme digestion reaction 4: the mixture of the fragmenting enzymes IV+ enzyme reaction buffer.
Example 3 preparation of a reaction solution for Joint ligation
The linker ligation reaction solution contained two parts: the linker ligase mixture and the linker ligation reaction buffer.
1. Preparing a joint ligase mixed solution:
component of the adaptor-ligase mixed solution 1 and final concentration of each component in the adaptor-ligase mixed solution 1: t4 DNA ligase (800 ng/. Mu.l), T4 PNK (40 ng/. Mu.l), tris-Ac (pH 8.0, 50 mM), tween 20 (V/V, 0.5%), DTT (1 mM), glycerol (V/V, 50%).
The components of the linker-ligase mixture 2 and the final concentration of each component in the linker-ligase mixture 2: t4 DNA ligase (800 ng/. Mu.l), T4 PNK (80 ng/. Mu.l), tris-Ac (pH 8.0, 50 mM), tween 20 (V/V, 0.5%), DTT (1 mM), glycerol (V/V, 50%).
2. Preparing a joint connection reaction buffer solution:
linker ligation reaction buffer: adapter Ligation Buffer in TransGen#KP201TransNGS DNA Library Prep Kit for Illumina was used.
3. Preparing a joint connection reaction liquid:
the joint connection reaction liquid is composed of a joint connection enzyme mixed liquid and a joint connection reaction buffer liquid:
joint connection reaction liquid 1: the joint ligase mixed solution 1+ joints are connected with a reaction buffer solution;
and (2) joint connection reaction liquid: the linker ligase mixture was 2+ linker ligation reaction buffer.
EXAMPLE 4 construction of DNA sequencing library
In this example, heLa cell gDNA was used as a template to initiate the castingThe amounts of introduction were 100pg, 1ng, 10ng, 100ng and 1. Mu.g, respectively, gDNA was fragmented, end-repaired and 3' tailing was performed using the different cleavage reaction solutions of example 1, followed by linker ligation using the different linker ligation reaction solutions of example 2 (linker #KP201DNA Library Prep Kit for Illumina->Adapter for />Or Adapter in KI 401), then the adaptor ligation product was purified using TransGen#EC401 MagicPure Size Selection DNA Beads using TransGen#KP201 +.>DNA Library Prep Kit for/>Is->Library Amplification SuperMix (2X) and i5/i7 Primer in TransGen#KI251 PCR amplified enrichment of the purified ligation products, and finally the construction of the DNA sequencing library was completed.
Fragmentation of HeLa cell gDNA, end repair and 3' addition of dA tail
The reaction system of the fragmentation, end repair and 3' dA tail addition of the HeLa cell gDNA is shown in Table 1, wherein the formulation of the mixed solution of the fragmenting enzymes adopts one of the mixed solutions I to III of example 1, the enzyme digestion reaction buffer of example 1 is adopted, and the input amount of the HeLa cell gDNA (hereinafter abbreviated as DNA) is X (corresponding to 100pg, 1ng, 10ng, 100ng and 1. Mu.g, respectively).
TABLE 1 reaction System for fragmentation, terminal repair and 3' dA tail addition
And (3) blowing and sucking by using a pipette, and then incubating for 12 minutes at 37 ℃ and 30 minutes at 65 ℃ according to the reaction conditions to perform enzyme digestion (fragmentation), end repair and 3' addition of A tail of the DNA, so as to obtain a fragmented product.
2. 5' phosphorylation, linker ligation and purification of fragmented products
After fragmentation, end repair and 3 'dA tail addition, 5' phosphorylation, linker ligation and purification of the fragmented product is required.
Using TransGen#KP201Adapter Dilution Buffer pair #KP201 +.in DNA Library Prep Kit for Illumina>DNA Library Prep Kit for Illumina->Adapter for/>The linker was diluted to give a linker of a corresponding concentration, and the specific dilution ratios are shown in table 2.
TABLE 2 dilution ratio of adaptors at different DNA input amounts
| DNA input amount | Dilution of joint | Concentration of the corresponding linker after dilution |
| 100ng＜x≤1μg | Not diluting | 16μM |
| 25ng＜x≤100ng | Diluted 2 times | 8μM |
| 5ng＜x≤25ng | Diluted 10 times | 1.6μM |
| 100pg≤x≤5ng | Diluted 25 times | 0.6μM |
The 5' phosphorylation and linker ligation reaction system is shown in Table 3, wherein the formula of the linker ligase mixture was one of the linker ligase mixtures 1 and 2 of example 2, and the linker ligation reaction buffer was the linker ligation reaction buffer of example 2.
Table 3 5' phosphorylation and linker ligation reaction System
And (3) blowing and sucking the mixture uniformly by using a pipette, and then incubating the mixture for 15 minutes at 25 ℃ according to the reaction condition to carry out 5' phosphorylation and linker ligation reaction (long linker recommended 20 ℃ ligation reaction for 15 minutes) to obtain a phosphorylated and linker ligation product.
The phosphorylated and adaptor-ligated product was purified using 80. Mu.L TransGen#EC401 MagicPure Size Selection DNA Beads, eluting at a volume of 20/23. Mu.L, to give a purified phosphorylated and adaptor-ligated product.
3. PCR amplification enrichment of purified adaptor ligation products
Using TransGen#KP201Library Amplification SuperMix (library amplification reaction solution) and TransGen#KI251 ∈ ->UDI Primers(96)Kit for/>(i 5/i7 Pirmer) PCR amplification enrichment was performed on the purified adaptor-ligated product, and the PCR amplification enrichment system is shown in Table 4.
TABLE 4 PCR amplification enrichment System
| Component (A) | Dosage of |
| Phosphorylation and linker ligation products | 20μL |
| Library amplification reaction solution | 25μL |
| i5 Primer | 2.5μL |
| i7 Primer | 2.5μL |
And (3) blowing and sucking uniformly by using a pipette, and then carrying out PCR amplification enrichment according to the reaction conditions to obtain an amplification product.
The PCR amplification conditions were as follows:
(the number of amplification cycles for different amounts of DNA added are shown in Table 5)
TABLE 5 amplification cycle number of different DNA input amounts
| DNA input amount | Amplification cycle number X |
| 100pg | 17 |
| 1ng | 15 |
| 10ng | 9 |
| 100ng | 4 |
| 1μg | 2 |
After amplification, the amplified product was purified using 50 μl of TransGen#EC401 MagicPure Size Selection DNA Beads to obtain a DNA sequencing library; quantification of DNA sequencing library yield using Qubit; the length distribution of the DNA sequencing library was examined using the Agilent 2100 DNA 1000 chip, and the results are shown in Table 6 and FIGS. 1-6. It can be seen that each set of cleavage reaction solution and linker ligation reaction solution combination can achieve efficient cleavage (fragmentation), end repair, 3 'dA tail addition, 5' phosphorylation and linker ligation of DNA of different input amounts.
Different combined list:
combination 1: the enzyme digestion reaction liquid 1+ connector is connected with the reaction liquid 1; combination 2: the enzyme digestion reaction liquid 2+ connector is connected with the reaction liquid 1; combination 3: the enzyme digestion reaction liquid 3+ connector is connected with the reaction liquid 1; combination 4: the enzyme digestion reaction liquid 4+ connector is connected with the reaction liquid 1; combination 5: the enzyme digestion reaction liquid 1+ connector is connected with the reaction liquid 2; combination 6: the enzyme digestion reaction liquid 2+ connector is connected with the reaction liquid 2; combination 7: the enzyme digestion reaction liquid 3+ connector is connected with the reaction liquid 2; combination 8: the enzyme digestion reaction liquid 4+ connector is connected with the reaction liquid 2.
TABLE 6 yield of libraries constructed from different combinations (Unit: ng)
| DNA input amount | Combination 1 | Combination 2 | Combination 3 | Combination 5 | Combination 6 | Combination 7 |
| 100pg | 676.3 | 777.7 | 894.4 | 689.8 | 786.5 | 914.7 |
| 1ng | 882.5 | 1017.5 | 1173.2 | 917.8 | 1028.1 | 1197.8 |
| 10ng | 1011.2 | 1167.9 | 1349.0 | 1071.9 | 1180.1 | 1377.1 |
| 100ng | 1355.2 | 1568.0 | 1814.1 | 1463.6 | 1584.2 | 1852.0 |
| 1μg | 1566.4 | 1815.5 | 2104.1 | 1723.0 | 1834.3 | 2147.9 |
* Wherein, combination 4 and combination 8 failed to build a pool, there was no data.
From the analysis it is known that: combination 4 and combination 8 failed to build the library. The DNA sequencing libraries were successfully constructed from combinations 1 to 3 and from combinations 5 to 7, and the peak patterns were not greatly different, which indicates that the addition of the recombinant topoisomerase of the invention had little effect on the peak patterns; the yield of the DNA sequencing library constructed in combination 1 relative to combination 5, combination 2 relative to combination 6 and combination 3 relative to combination 7 was slightly increased, indicating that the yield of the library was slightly increased with increasing T4 PNK; the lower yields of combination 1 relative to combinations 2 and 3 and combination 5 relative to combinations 6 and 7 indicate that the addition of the recombinant topoisomerase of the invention provides a significant increase in the yield of the DNA sequencing library.
Example 5 GC distribution analysis
From the DNA sequencing library yields and peak patterns of example 4, the yields of the DNA sequencing libraries constructed in combination 5, combination 6 and combination 7 were higher than those in combination 1, combination 2 and combination 3, respectively, so that the DNA sequencing libraries constructed in combination 5, combination 6 and combination 7 were sorted (0.65×+0.2×) using the transgen#ec401 MagicPure Size Selection DNA Beads and sent to a third party sequencing company for sequencing and analysis, and the analysis results are shown in table 7.
TABLE 7 sequencing data volume and Mass analysis results
The GC content distribution of the genome and the GC distribution in the alignment result were counted using Picard (version 1.119, parameter: collectGcBIas Metrics. Jar) and plotted to determine whether the sequencing data had a GC bias (generally, the portions with low and high GC content had a certain GC bias, the closer the relative coverage was to 1, the smaller the GC bias).
The GC content distribution and depth distribution after library construction using combinations 5, 6, 7 for different DNA input amounts are shown in FIG. 7A-E (the abscissa represents GC content (genome calculated with 100bp as window), and the ordinate represents relative coverage of specific base content (left) and window ratio of occupied genome (right)).
As can be seen from fig. 7, the GC distribution of the high GC region is more balanced after the recombinant topoisomerase of example 1 of the present invention was added to combinations 6 and 7 relative to combination 5.
In conclusion, the addition of the recombinant topoisomerase does not affect the digestion effect of the digestion reaction liquid on DNA, but obviously improves the yield of the constructed DNA sequencing library, and obviously improves the deviation of GC distribution in sequencing data analysis.
Therefore, the addition of the recombinant topoisomerase improves the library construction quality of the DNA sequencing library construction by the enzyme digestion method, and greatly ensures the subsequent sequencing data analysis.
It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.
SEQUENCE LISTING
<110> Beijing full gold biotechnology Co., ltd
<120> a recombinant topoisomerase and its use in the construction of sequencing libraries
<130> JLP21I1306
<160> 6
<170> PatentIn version 3.5
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Met Gly Lys Ala Leu Val Ile Val Glu Ser Pro Ala Lys Ala Lys Thr
1 5 10 15
Ile Asn Lys Tyr Leu Gly Ser Asp Tyr Val Val Lys Ser Ser Val Gly
20 25 30
His Ile Arg Asp Leu Pro Thr Ser Gly Ser Ala Ala Lys Ala Ser Ala
35 40 45
Asp Ser Thr Ser Thr Lys Thr Ala Lys Lys Pro Lys Lys Asp Glu Arg
50 55 60
Gly Ala Leu Val Asn Arg Met Gly Val Asp Pro Trp His Asn Trp Glu
65 70 75 80
Ala His Tyr Glu Val Leu Pro Gly Ala Glu Lys Val Val Ser Glu Leu
85 90 95
Ala Gln Leu Ala Glu Lys Ala Asp His Ile Tyr Leu Ala Thr Asp Leu
100 105 110
Asp Arg Glu Gly Glu Ala Ile Ala Trp His Leu Arg Glu Val Ile Gly
115 120 125
Gly Asp Asp Ala Arg Tyr Ser Arg Val Val Phe Asn Glu Ile Thr Ala
130 135 140
Asn Ala Ile Arg Gln Ala Phe Asn Lys Pro Gly Glu Leu Asn Ile Asp
145 150 155 160
Arg Val Asn Ala Gln Gln Ala Arg Arg Phe Met Asp Arg Val Val Gly
165 170 175
Tyr Met Val Ser Pro Leu Leu Trp Lys Lys Ile Ala Arg Gly Leu Ser
180 185 190
Ala Gly Arg Val Gln Ser Val Ala Val Arg Leu Val Val Glu Arg Glu
195 200 205
Arg Glu Ile Lys Ala Phe Val Pro Glu Glu Phe Trp Glu Val Asp Ala
210 215 220
Ser Thr Thr Thr Pro Ser Gly Glu Ala Leu Ala Leu Gln Val Thr His
225 230 235 240
Gln Asn Asp Arg Pro Phe Arg Pro Val Asn Lys Glu Gln Thr Gln Ala
245 250 255
Ala Val Ser Leu Leu Glu Lys Ala Arg Tyr Ser Val Leu Glu Arg Glu
260 265 270
Asp Lys Pro Thr Thr Ser Lys Pro Gly Ala Pro Phe Ile Thr Ser Thr
275 280 285
Leu Gln Gln Ala Ala Ser Thr Arg Leu Gly Phe Gly Val Lys Lys Thr
290 295 300
Met Met Met Ala Gln Arg Leu Tyr Glu Ala Gly Tyr Ile Thr Tyr Met
305 310 315 320
Arg Thr Asp Ser Thr Asn Leu Ser Gln Asp Ala Val Asn Met Val Arg
325 330 335
Gly Tyr Ile Ser Asp Asn Phe Gly Lys Lys Tyr Lys Pro Glu Ser Pro
340 345 350
Asn Gln Tyr Ala Ser Lys Glu Asn Ser Gln Glu Ala His Glu Ala Ile
355 360 365
Arg Pro Ser Asp Val Asn Val Met Ala Glu Ser Leu Lys Asp Met Glu
370 375 380
Ala Asp Ala Gln Lys Leu Tyr Gln Leu Ile Trp Arg Gln Phe Val Ala
385 390 395 400
Cys Gln Met Thr Pro Ala Lys Tyr Asp Ser Thr Thr Leu Thr Val Glu
405 410 415
Ala Gly Asp Phe Arg Leu Lys Ala Arg Gly Arg Ile Leu Arg Phe Asp
420 425 430
Gly Trp Thr Lys Val Met Pro Ala Leu Arg Lys Gly Asp Glu Asp Arg
435 440 445
Ile Leu Pro Ala Val Asn Lys Gly Asp Ala Leu Thr Leu Val Glu Leu
450 455 460
Thr Pro Ala Gln His Phe Thr Lys Pro Pro Ala Arg Phe Ser Glu Ala
465 470 475 480
Ser Leu Val Tyr Glu Leu Glu Lys Arg Gly Ile Gly Arg Pro Ser Thr
485 490 495
Tyr Ala Ser Ile Ile Ser Thr Ile Gln Asp Arg Gly Tyr Val Arg Val
500 505 510
Glu Asn Arg Arg Phe Tyr Ala Glu Lys Met Gly Glu Ile Val Thr Asp
515 520 525
Arg Leu Glu Glu Asn Phe Arg Glu Leu Met Asn Tyr Asp Phe Thr Ala
530 535 540
Gln Met Glu Asn Ser Leu Asp Gln Val Ala Asn His Glu Ala Glu Trp
545 550 555 560
Lys Ala Val Leu Asp His Phe Phe Ser Asp Phe Thr Gln Gln Leu Asp
565 570 575
Lys Ala Glu Lys Asp Pro Glu Glu Gly Gly Met Arg Pro Asn Gln Met
580 585 590
Val Leu Thr Ser Ile Asp Cys Pro Thr Cys Gly Arg Lys Met Gly Ile
595 600 605
Arg Thr Ala Ser Thr Gly Val Phe Leu Gly Cys Ser Gly Tyr Ala Leu
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Pro Pro Lys Glu Arg Cys Lys Thr Thr Ile Asn Leu Val Pro Glu Asn
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Glu Val Leu Asn Val Leu Glu Gly Glu Asp Ala Glu Thr Asn Ala Leu
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Arg Ala Lys Arg Arg Cys Pro Lys Cys Gly Thr Ala Met Asp Ser Tyr
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Leu Ile Asp Pro Lys Arg Lys Leu His Val Cys Gly Asn Asn Pro Thr
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Cys Asp Gly Tyr Glu Ile Glu Glu Gly Glu Phe Arg Ile Tyr Gly Tyr
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Asp Gly Pro Ile Val Glu Cys Glu Lys Cys Gly Ser Glu Met His Leu
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Lys Met Gly Arg Phe Gly Lys Tyr Met Ala Cys Thr Asn Glu Glu Cys
725 730 735
Lys Asn Thr Arg Lys Ile Leu Arg Asn Gly Glu Val Ala Pro Pro Lys
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Glu Asp Pro Val Pro Leu Pro Glu Leu Pro Cys Glu Lys Ser Asp Ala
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Tyr Phe Val Leu Arg Asp Gly Ala Ala Gly Val Phe Leu Ala Ala Asn
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Thr Phe Pro Lys Ser Arg Glu Thr Arg Ala Pro Leu Val Glu Glu Leu
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Tyr Arg Phe Arg Asp Arg Leu Pro Glu Lys Leu Arg Tyr Leu Ala Asp
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Ala Pro Gln Gln Asp Pro Glu Gly Asn Lys Thr Met Val Arg Phe Ser
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Arg Lys Thr Lys Gln Gln Tyr Val Ser Ser Glu Lys Asp Gly Lys Ala
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Met Glu Ser Leu Ser Glu Gly Thr Thr Ala Gly Tyr Gln Gln Ile His
1 5 10 15
Asp Gly Ile Ile His Leu Val Asp Ser Ala Arg Thr Glu Thr Val Arg
20 25 30
Ser Val Asn Ala Leu Met Thr Ala Thr Tyr Gln Glu Ile Gly Arg Arg
35 40 45
Ile Val Gln Phe Glu Gln Gly Gly Glu Ala Arg Ala Ala Tyr Gly Ala
50 55 60
Gln Leu Ile Lys Arg Leu Ser Lys Asp Leu Cys Leu Arg Tyr Lys Arg
65 70 75 80
Gly Phe Ser Ala Lys Asn Leu Arg Gln Met Arg Leu Phe Tyr Leu Phe
85 90 95
Phe Gln His Val Glu Ile His Gln Thr Met Ser Gly Glu Leu Thr Pro
100 105 110
Leu Gly Ile Pro Gln Thr Pro Ser Ala Glu Phe Pro Ser Ala Lys Ile
115 120 125
Trp Gln Thr Leu Ser Ala Lys Ser Phe Pro Leu Pro Arg Ser Thr Tyr
130 135 140
Val Arg Leu Leu Ser Val Lys Asn Ala Asp Ala Arg Ser Phe Tyr Glu
145 150 155 160
Lys Glu Thr Leu Arg Cys Gly Trp Ser Val Arg Gln Leu Glu Arg Gln
165 170 175
Ile Ala Thr Gln Phe Tyr Glu Arg Thr Leu Leu Ser His Asp Ala Ser
180 185 190
Ala Met Leu Gln Gln His Ala Pro Ala Glu Thr His Ile Leu Pro Gln
195 200 205
Gln Ala Ile Arg Asp Pro Phe Val Leu Glu Phe Leu Glu Leu Lys Asp
210 215 220
Glu Phe Ser Glu Ser Asp Phe Glu Glu Ala Leu Ile Asn His Leu Met
225 230 235 240
Asp Phe Met Leu Glu Leu Gly Asp Asp Phe Ala Phe Val Gly Arg Gln
245 250 255
Arg Arg Leu Arg Ile Asp Asp Asn Trp Phe Arg Val Asp Leu Leu Phe
260 265 270
Phe His Arg Arg Leu Arg Cys Leu Leu Ile Val Asp Leu Lys Val Gly
275 280 285
Lys Phe Ser Tyr Ser Asp Ala Gly Gln Met Asn Met Tyr Leu Asn Tyr
290 295 300
Ala Lys Glu His Trp Thr Leu Pro Asp Glu Asn Pro Pro Ile Gly Leu
305 310 315 320
Val Leu Cys Ala Glu Lys Gly Ala Gly Glu Ala His Tyr Ala Leu Ala
325 330 335
Gly Leu Pro Asn Thr Val Leu Ala Ser Glu Tyr Lys Met Gln Leu Pro
340 345 350
Asp Glu Lys Arg Leu Ala Asp Gly Leu Val Arg Thr Gln Ala Val Leu
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Glu Glu Gly Tyr Arg Arg Arg
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<210> 3
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<213> Artificial sequence (Artificial Sequence)
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Met Cys Leu Pro Phe Gln Gln Asp Phe Ile Glu Thr Asp Leu Ile Leu
1 5 10 15
Leu Met Asn Tyr Ile Cys Ser Lys Gln Ala Ile Leu Ser Ser Lys Phe
20 25 30
Thr Pro Cys Cys Glu Leu Pro Glu Pro Phe Arg Gly Glu Cys Ile Val
35 40 45
Asn Ser Glu Asn Asp Asn Lys Pro Asp Leu Ser Ser Leu Pro His Arg
50 55 60
Arg Tyr Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Leu Ala Leu Pro
65 70 75 80
Val Ile Leu Arg Val Asp Thr Val Tyr Gln Asn Leu Leu Gly Lys Cys
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Cys Lys Leu Glu Asn Pro Leu Glu Cys Cys Ser His Gly Gln Gly Met
100 105 110
Phe Gln Arg Val Val Arg Glu Ser His Glu Arg Val Lys Asn Gln Cys
115 120 125
Asp Leu Arg Lys Lys Leu Gly Asp Ser Asn Phe His Asp Arg Leu Ile
130 135 140
Val Leu Tyr Thr Lys His Ala Pro Gln Leu Ser Ala Gln Glu Leu Ile
145 150 155 160
Val Leu Thr Lys Asn Met Ala Ala Ala Ala Thr Lys Cys Cys Pro Leu
165 170 175
Asn Gln Glu His Gln Phe Val Cys Met Glu Asp Ser Ala Lys Leu Ile
180 185 190
Leu Gly Ala Leu Cys Arg Arg His Gln Ala Glu Pro Ile Asn Ala Ala
195 200 205
Val Gly His Cys Cys Asp Asp Ser Tyr Ala Phe Arg Lys Pro Cys Phe
210 215 220
Asp Asp Leu Gln Val Asp Gly Thr Tyr Ile Ser Ser Pro Leu Ser Cys
225 230 235 240
Asp Gln Val Ile Asp Leu Lys Glu Asp Leu Cys Lys Ala Gln Glu Glu
245 250 255
Glu Leu Glu Thr Glu Lys Gln Lys Leu Leu Ser Asn Leu Val Lys Glu
260 265 270
Lys Leu Tyr Ala Ala Glu Met Gln Phe Gln Pro Ile Thr Val Asp Phe
275 280 285
Ala His Leu Val Glu Thr Cys Cys Gln Ala Glu Lys Ser Glu Thr Cys
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Phe Gln Glu Gln Val Ser Leu Phe Pro Cys Leu Phe Ser
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<210> 4
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<212> PRT
<213> Artificial sequence (Artificial Sequence)
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Met Ala Ser Val Asn Lys Val Ile Ile Val Gly Asn Leu Gly Arg Asp
1 5 10 15
Pro Glu Thr Arg Ser Phe Pro Asn Gly Asp Glu Val Thr Asn Ile Thr
20 25 30
Ile Ala Thr Thr Asp Arg Tyr Lys Asp Lys Thr Ser Gly Glu Ala Lys
35 40 45
Glu Ile Thr Glu Trp His Arg Val Ser Phe Phe Gly Arg Leu Ala Glu
50 55 60
Ile Ala His Gln Tyr Leu Arg Lys Gly Ser Gln Val Tyr Val Glu Gly
65 70 75 80
Ser Leu Arg Thr Arg Lys Trp Thr Asp Lys Asp Gly Ile Glu Lys Tyr
85 90 95
Thr Thr Glu Ile Arg Ala Asp Ser Met Gln Cys Leu Gly Ser Arg Gln
100 105 110
Gly Met Gly Gly Ala Gln Thr Gly Asp Glu Gly Gly Gly Gly Tyr Glu
115 120 125
Pro Ala Ala Arg Ala Ala Pro Ala Ala Arg Pro Ala Met Val Ala Ala
130 135 140
Pro Arg Gln Ala Pro Ala Pro Ala Ala Lys Thr Ala Ser Gly Phe Asp
145 150 155 160
Asp Met Asp Asp Asp Ile Pro Phe
165
<210> 5
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<212> PRT
<213> Artificial sequence (Artificial Sequence)
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Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly Ala Phe Val Gly
1 5 10 15
Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp Leu Leu Ala Leu
20 25 30
Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro Glu Pro Tyr Lys
35 40 45
Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu Ala Lys Asp Leu
50 55 60
Ser Val Leu Ala Leu Arg Glu Gly Leu Gln Leu Pro Pro Gly Asp Asp
65 70 75 80
Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr Thr Pro Glu
85 90 95
Gly Val Ala Arg Arg Tyr Glu Gly Glu Trp Thr Glu Glu Ala Gly Glu
100 105 110
Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu Trp Gly Arg Leu
115 120 125
Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu Val Glu Arg Pro
130 135 140
Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly Val Arg Leu Asp
145 150 155 160
Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala Glu Glu Ile Ala
165 170 175
Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His Pro Phe Asn Leu
180 185 190
Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp Glu Leu Gly Leu
195 200 205
Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg Ser Thr Ser Ala
210 215 220
Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile Val Glu Lys Ile
225 230 235 240
Leu Gln Tyr Arg Gly Leu Thr Lys Leu Lys Ser Thr Tyr Ile Asp Pro
245 250 255
Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu His Thr Arg Phe
260 265 270
Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp Pro Asn
275 280 285
Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln Arg Ile Arg Arg
290 295 300
Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala Leu Asp Tyr Ser
305 310 315 320
Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly Asp Glu Asn Leu
325 330 335
Ile Arg Val Phe Gln Gly Gly Arg Asp Ile His Thr Glu Thr Ala Ser
340 345 350
Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met Arg Arg
355 360 365
Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly Met Ser Ala His
370 375 380
Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu Ala Gln Ala Phe
385 390 395 400
Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg Ala Trp Ile Glu
405 410 415
Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val Glu Thr Leu Phe
420 425 430
Gly Arg Arg Arg Tyr Val Pro Asp Leu Gly Ala Arg Val Lys Ser Val
435 440 445
Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro Val Gln Gly Thr
450 455 460
Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu Phe Pro Arg Leu
465 470 475 480
Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His Asp Glu Leu Val
485 490 495
Leu Glu Ala Pro Lys Glu Arg Ala Gln Ala Val Ala Arg Leu Ala Lys
500 505 510
Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro Leu Glu Val Glu
515 520 525
Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys
530 535
<210> 6
<211> 865
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Met Gly Lys Ala Leu Val Ile Val Glu Ser Pro Ala Lys Ala Lys Thr
1 5 10 15
Ile Asn Lys Tyr Leu Gly Ser Asp Tyr Val Val Lys Ser Ser Val Gly
20 25 30
His Ile Arg Asp Leu Pro Thr Ser Gly Ser Ala Ala Lys Lys Ser Ala
35 40 45
Asp Ser Thr Ser Thr Lys Thr Ala Lys Lys Pro Lys Lys Asp Glu Arg
50 55 60
Gly Ala Leu Val Asn Arg Met Gly Val Asp Pro Trp His Asn Trp Glu
65 70 75 80
Ala His Tyr Glu Val Leu Pro Gly Lys Glu Lys Val Val Ser Glu Leu
85 90 95
Lys Gln Leu Ala Glu Lys Ala Asp His Ile Tyr Leu Ala Thr Asp Leu
100 105 110
Asp Arg Glu Gly Glu Ala Ile Ala Trp His Leu Arg Glu Val Ile Gly
115 120 125
Gly Asp Asp Ala Arg Tyr Ser Arg Val Val Phe Asn Glu Ile Thr Lys
130 135 140
Asn Ala Ile Arg Gln Ala Phe Asn Lys Pro Gly Glu Leu Asn Ile Asp
145 150 155 160
Arg Val Asn Ala Gln Gln Ala Arg Arg Phe Met Asp Arg Val Val Gly
165 170 175
Tyr Met Val Ser Pro Leu Leu Trp Lys Lys Ile Ala Arg Gly Leu Ser
180 185 190
Ala Gly Arg Val Gln Ser Val Ala Val Arg Leu Val Val Glu Arg Glu
195 200 205
Arg Glu Ile Lys Ala Phe Val Pro Glu Glu Phe Trp Glu Val Asp Ala
210 215 220
Ser Thr Thr Thr Pro Ser Gly Glu Ala Leu Ala Leu Gln Val Thr His
225 230 235 240
Gln Asn Asp Lys Pro Phe Arg Pro Val Asn Lys Glu Gln Thr Gln Ala
245 250 255
Ala Val Ser Leu Leu Glu Lys Ala Arg Tyr Ser Val Leu Glu Arg Glu
260 265 270
Asp Lys Pro Thr Thr Ser Lys Pro Gly Ala Pro Phe Ile Thr Ser Thr
275 280 285
Leu Gln Gln Ala Ala Ser Thr Arg Leu Gly Phe Gly Val Lys Lys Thr
290 295 300
Met Met Met Ala Gln Arg Leu Tyr Glu Ala Gly Tyr Ile Thr Tyr Met
305 310 315 320
Arg Thr Asp Ser Thr Asn Leu Ser Gln Asp Ala Val Asn Met Val Arg
325 330 335
Gly Tyr Ile Ser Asp Asn Phe Gly Lys Lys Tyr Leu Pro Glu Ser Pro
340 345 350
Asn Gln Tyr Ala Ser Lys Glu Asn Ser Gln Glu Ala His Glu Ala Ile
355 360 365
Arg Pro Ser Asp Val Asn Val Met Ala Glu Ser Leu Lys Asp Met Glu
370 375 380
Ala Asp Ala Gln Lys Leu Tyr Gln Leu Ile Trp Arg Gln Phe Val Ala
385 390 395 400
Cys Gln Met Thr Pro Ala Lys Tyr Asp Ser Thr Thr Leu Thr Val Gly
405 410 415
Ala Gly Asp Phe Arg Leu Lys Ala Arg Gly Arg Ile Leu Arg Phe Asp
420 425 430
Gly Trp Thr Lys Val Met Pro Ala Leu Arg Lys Gly Asp Glu Asp Arg
435 440 445
Ile Leu Pro Ala Val Asn Lys Gly Asp Ala Leu Thr Leu Val Glu Leu
450 455 460
Thr Pro Ala Gln His Phe Thr Lys Pro Pro Ala Arg Phe Ser Glu Ala
465 470 475 480
Ser Leu Val Lys Glu Leu Glu Lys Arg Gly Ile Gly Arg Pro Ser Thr
485 490 495
Tyr Ala Ser Ile Ile Ser Thr Ile Gln Asp Arg Gly Tyr Val Arg Val
500 505 510
Glu Asn Arg Arg Phe Tyr Ala Glu Lys Met Gly Glu Ile Val Thr Asp
515 520 525
Arg Leu Glu Glu Asn Phe Arg Glu Leu Met Asn Tyr Asp Phe Thr Ala
530 535 540
Gln Met Glu Asn Ser Leu Asp Gln Val Ala Asn His Glu Ala Glu Trp
545 550 555 560
Lys Ala Val Leu Asp His Phe Phe Ser Asp Phe Thr Gln Gln Leu Asp
565 570 575
Lys Ala Glu Lys Asp Pro Glu Glu Gly Gly Met Arg Pro Asn Gln Met
580 585 590
Val Leu Thr Ser Ile Asp Cys Pro Thr Cys Gly Arg Lys Met Gly Ile
595 600 605
Arg Thr Ala Ser Thr Gly Val Phe Leu Gly Cys Ser Gly Tyr Ala Leu
610 615 620
Pro Pro Lys Glu Arg Cys Lys Thr Thr Ile Asn Leu Val Pro Glu Asn
625 630 635 640
Glu Val Leu Asn Val Leu Glu Gly Glu Asp Ala Glu Thr Asn Ala Leu
645 650 655
Arg Ala Lys Arg Arg Cys Pro Lys Cys Gly Thr Ala Met Asp Ser Tyr
660 665 670
Leu Ile Asp Pro Lys Arg Lys Leu His Val Cys Gly Asn Asn Pro Thr
675 680 685
Cys Asp Gly Tyr Glu Ile Glu Glu Gly Glu Phe Arg Ile Lys Gly Tyr
690 695 700
Asp Gly Pro Ile Val Glu Cys Glu Lys Cys Gly Ser Glu Met His Leu
705 710 715 720
Lys Met Gly Arg Phe Gly Lys Tyr Met Ala Cys Thr Asn Glu Glu Cys
725 730 735
Lys Asn Thr Arg Lys Ile Leu Arg Asn Gly Glu Val Ala Pro Pro Lys
740 745 750
Glu Asp Pro Val Pro Leu Pro Glu Leu Pro Cys Glu Lys Ser Asp Ala
755 760 765
Tyr Phe Val Leu Arg Asp Gly Ala Ala Gly Val Phe Leu Ala Ala Asn
770 775 780
Thr Phe Pro Lys Ser Arg Glu Thr Arg Ala Pro Leu Val Glu Glu Leu
785 790 795 800
Tyr Arg Phe Arg Asp Arg Leu Pro Glu Lys Leu Arg Tyr Leu Ala Asp
805 810 815
Ala Pro Gln Gln Asp Pro Glu Gly Asn Lys Thr Met Val Arg Phe Ser
820 825 830
Arg Lys Thr Lys Gln Gln Tyr Val Ser Ser Glu Lys Asp Gly Lys Ala
835 840 845
Thr Gly Trp Ser Ala Phe Tyr Val Asp Gly Lys Trp Val Glu Gly Lys
850 855 860
Lys
865
Claims (20)
1. A recombinant topoisomerase is characterized in that the amino acid sequence of the recombinant topoisomerase is shown as SEQ ID NO. 1.
2. An enzyme cleavage reaction solution, characterized in that the enzyme cleavage reaction solution comprises a mixed solution of fragmenting enzymes, which comprises the recombinant topoisomerase according to claim 1.
3. The cleavage reaction liquid according to claim 2, wherein the mixed solution of fragmenting enzymes further comprises endonuclease and DNA polymerase.
4. The cleavage reaction solution according to claim 3, wherein the endonuclease comprises any one or a combination of two of a double-stranded DNA nuclease and an ATP-dependent nuclease; the DNA polymerase includes any one or a combination of two of low temperature DNA polymerase and thermostable DNA polymerase.
5. The cleavage reaction solution according to claim 4, characterized in that the low-temperature DNA polymerase comprises any one or a combination of two of Phi29DNA polymerase and T4 DNA polymerase large fragment Klenow; the heat-resistant DNA polymerase comprises any one or the combination of two of Bst II DNA polymerase and Taq DNA polymerase large fragment Klenow.
6. The cleavage reaction solution according to claim 3, wherein the mixed solution of the fragmenting enzymes further comprises an auxiliary protein.
7. The cleavage reaction solution according to claim 6, wherein the accessory protein comprises any one or a combination of two of recombinant albumin and SSB.
8. The cleavage reaction liquid of claim 2, further comprising a cleavage reaction buffer comprising a metal cation, a substrate, and a buffer medium.
9. The cleavage reaction solution according to claim 8, characterized in that the metal cation comprises Mg 2+ 、K + And NH 3+ Any one or a combination of a plurality of the above; the substrate comprises any one or more of dNTPs, dATP and ATP; the buffer medium comprises a combination of any one or more of 2-morpholinoethanesulfonic acid, acetic acid and tris.
10. The cleavage reaction solution according to claim 2, characterized in that the mixture of fragmenting enzymes consists of the recombinant topoisomerase of claim 1, ATP dependent nuclease, phi29DNA polymerase, bst II DNA polymerase, SSB, recombinant albumin, dsDNase, tris-HCl, KCl, tween, DTT and glycerol.
11. The cleavage reaction solution of claim 10, further comprising a cleavage reaction buffer consisting of Tris-acetic acid, mgCl2, KAc, dNPTs, dATP, and ATP.
12. A kit comprising the recombinant topoisomerase of claim 1 or the cleavage reaction solution of any one of claims 2-11.
13. The kit of claim 12, further comprising a linker ligation reaction solution comprising a linker ligase mixture and a linker ligation reaction buffer.
14. The kit of claim 13, wherein the adaptor ligase mixture comprises a polynucleotide kinase and a ligase.
15. The kit of claim 14, wherein the polynucleotide kinase is a T4 polynucleotide kinase and the ligase is a T4 DNA ligase.
16. Use of the recombinant topoisomerase of claim 1, the cleavage reaction solution of any one of claims 2-11, or the kit of any one of claims 12-15 in the construction of a sequencing library or in the preparation of reagents for the construction of a sequencing library.
17. A method of constructing a sequencing library, the method comprising:
fragmenting target DNA, repairing the tail and adding 3' A tail by using the enzyme digestion reaction liquid containing the recombinant topoisomerase of claim 1, the enzyme digestion reaction liquid of any one of claims 2-11 or the enzyme digestion reaction liquid in the kit of any one of claims 12-15 to obtain fragmented products;
5' phosphorylation and linker ligation reaction are carried out on the fragmented product to obtain a phosphorylated and linker ligation product;
and carrying out PCR amplification enrichment on the phosphorylation and joint connection products to obtain a sequencing library.
18. The method of claim 17, wherein the conditions for fragmenting, end repair and 3' addition of a tail are incubation at 32 ℃ or 37 ℃ for 5-30 minutes followed by incubation at 65 ℃ for 20-30 minutes.
19. The method according to claim 17, wherein the 5 'phosphorylation and ligation reaction is performed in the ligation reaction solution of the kit according to any one of claims 12 to 15, and the reaction conditions of the 5' phosphorylation and ligation reaction are 25℃for 15 to 20 minutes.
20. The method according to claim 17, wherein the target DNA is added in an amount of 100 to pg. Mu.g.
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