A composition and its application in nucleic acid purification

Abstract

The present application provides a composition and its application in nucleic acid purification, belonging to the field of biotechnology. The composition can purify nucleic acids and effectively increase the proportion of 150-300bp small fragment libraries in library products.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and more particularly to an application of a composition in nucleic acid purification. The composition can purify nucleic acids and effectively increase the proportion of 150-300 bp small fragment libraries in library products. Background Art

[0002] Chromatin immunoprecipitation (ChIP) is a widely used method to study protein-DNA interactions, and is usually used to study transcription factor binding sites or histone-specific modification sites. Combining ChIP with massively parallel DNA sequencing technology allows researchers to accurately map the global DNA binding sites of proteins of interest. The basic process of ChIP is: (1) Cross-linking: using formaldehyde to fix tissues or cells to allow DNA and proteins to bind tightly; (2) Fragmentation: this process destroys chromatin and ultimately obtains DNA fragments and protein complexes for ChIP analysis; (3) Chromatin immunoprecipitation: by adding antibodies against the target protein, the target protein-DNA complex is bound; (4) DNA recovery and purification: recover and purify the enriched DNA fragments, and analyze the DNA sequence information specifically bound to the target protein through downstream detection technology (quantitative PCR, gene chip, sequencing, etc.) (Park PJ. ChIP-seq: advantages and challenges of a maturing technology. [J]. Nature Reviews Genetics, 2009, 10 (10): 669.) Figure 1 ). However, because ChIP technology requires formaldehyde cross-linking of tissues or cells and then DNA fragmentation, this technology requires an extremely high initial cell input (millions), and it is very likely that excessive formaldehyde cross-linking will cause false positives in the experimental results.

[0003] In order to make up for the technical defects of ChIP, researchers have developed CUT&Run (Cleavage Under Targets & Release Using Nuclease) technology and CUT&Tag (Cleavage Under Targets & Tagmentation) technology through continuous updating and optimization. CUT&Tag and CUT&Run are technologies for studying the interaction between proteins and DNA in living cells (Henikoff S, Skene P J. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites [J]. eLife, 6, (2017-01-06), 2017, 6; Kaya-Okur HS, Wu SJ, Codomo CA, et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells [J]. Nature Communications, 2019, 10 (1): 1930.), which enrich Tn5 transposase or Mnase nuclease through antibodies and specifically cut the DNA near the target protein. Then, by constructing and sequencing the library of the cleaved and labeled DNA, the global DNA binding site of the target protein can be mapped ( Figure 2 ). Because this technology does not require cross-linking and physical disruption of DNA, it alleviates the shortcomings of ChIP technology such as false positives and high cell input to a certain extent.

[0004] Although CUT&Tag and Cut&Run technologies are significantly improved compared to ChIP, CUT&Tag technology uses PA / G-Tn5 enzyme for genome cutting and fragmentation. The Tn5 enzyme itself has a large molecular weight of about 60KD, which makes it difficult to cut in the NDR region (nucleosome depleted region) at the same time. Therefore, it is difficult for CUT&Tag technology to enrich the signal of the NDR region alone. Cut&Run technology uses PA / G-MNase, and the molecular weight of the entire fusion enzyme is only 20KD. It can effectively enter the NDR region for cutting, but the current Cut&Run technology has weak performance in the recovery and library construction of small fragments, resulting in that Cut&Run still cannot recover small fragment information in the NDR region very well. Most transcription factors (TF: transcription factor) are bound to the NDR region ( Figure 3 ), so it is difficult to directly study the binding information of transcription factors and DNA. Summary of the invention

[0005] The purpose of the present application is to provide a method for connecting nucleic acid fragments and connectors, which can be used to improve the connection efficiency of 150-300bp small fragments and connectors during the construction of the CUT&Run library, thereby increasing the proportion of 150-300bp small fragments in the library and the success rate of library construction, so as to solve the problems of low small fragment recovery rate and weak library construction performance in the prior art, and at the same time improve the cell input compatibility of the CUT&Run technology.

[0006] In a first aspect, the present application provides a method for connecting a nucleic acid fragment and a linker, the method comprising using Sso7d-T4 DNA ligase to connect the nucleic acid fragment and the linker.

[0007] In some embodiments, the nucleic acid fragment is a double-stranded nucleic acid, which includes but is not limited to double-stranded DNA, double-stranded RNA and / or DNA-RNA hybrid double strands.

[0008] In some embodiments, the 3' end of the nucleic acid fragment contains a protruding adenine deoxyribonucleotide. In some embodiments, the nucleic acid fragment is connected to the adapter by AT connection, and the AT connection known in the art is that the adenine of the adenine deoxyribonucleotide is complementary to the thymine of the thymine deoxyribonucleotide of the adapter, thereby completing the connection between the nucleic acid fragment and the adapter. In some embodiments, the adapter is a Y-type adapter or a bubble adapter. In one embodiment, the adapter is a long or short sequencing adapter of the illumina platform or a bubble adapter of the MGI platform of MGI.

[0009] In some embodiments, both ends of the nucleic acid fragment are blunt ends, which can be connected to a blunt-end adapter, such as the sequencing adapter of the Ion Torrent platform.

[0010] In some embodiments, the Sso7d-T4 DNA ligase is a fusion protein, which is prepared according to methods known to those skilled in the art, for example, by constructing a recombinant vector in vitro and expressing and purifying it. In some embodiments, the Sso7d coding sequence and the T4 DNA ligase coding sequence of the recombinant expression vector are known in the art, including wild-type and mutant forms thereof.

[0011] In some embodiments, the Sso7d-T4 DNA ligase is a complex, prepared according to methods known to those skilled in the art, such as by connecting Sso7d protein and T4 DNA ligase in vitro. In some embodiments, the Sso7d protein sequence and T4 DNA ligase sequence are known in the art, including wild-type and mutant forms thereof.

[0012] In some embodiments, the Sso7d-T4 DNA ligase comprises the amino acid sequence shown in SEQ ID NO.1.

[0013] SEQ ID NO.1

[0014] DPALRATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKKGTSGGGSGGGILKILNEIASIGSTKQKQAILEKNKDNELLKRVYCLTYSRGLQYYIKKWPKPGIATQSFGMLT LTDMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMDRDLECGASVSIANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLKEELIKMTAEAR QIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDEAKVIYKKYIDQGLEGI ILKNIDGLWENARSKNLYKFKEVIDVDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL

[0015] In some embodiments, the nucleic acid fragment size is 50-500bp, preferably 100-400bp, more preferably 150-300bp, including 150bp, 155bp, 160bp, 165bp, 170bp, 175bp, 180bp, 185bp, 190bp, 195bp, 200bp, 205bp, 210bp, 215bp, 220bp, 225bp, 230bp, 235bp, 240bp, 245bp, 250bp, 255bp, 260bp, 265bp, 270bp, 275bp, 280bp, 285bp, 290bp, 295bp or 300bp within the range.

[0016] In some embodiments, the concentration of Sso7d-T4 DNA ligase in the reaction system is 1-10 mM, for example, 1 mM, 2 mM, 3 mM, 3.2 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM; preferably 2-6 mM, more preferably 3-5 mM.

[0017] In a second aspect, the present application provides a method for constructing a nucleic acid library, the method comprising:

[0018] Collecting a nucleic acid sample, and shearing the nucleic acid sample to obtain fragmented nucleic acid;

[0019] Optionally, purifying the fragmented nucleic acid;

[0020] Performing end repair on the fragmented nucleic acid to obtain nucleic acid fragments;

[0021] Using the method provided in the first aspect of the present application, connecting the nucleic acid fragment and the adapter;

[0022] Optionally, purifying the ligation product;

[0023] Optionally, amplifying the ligation product;

[0024] Optionally, the amplified product is purified.

[0025] In some embodiments, the interruption includes but is not limited to fragmenting the nucleic acid sample using an enzymatic method, a chemical method, and / or a mechanical method. In some embodiments, the enzymatic method is, for example, enzymatic cleavage using micrococcal nuclease (MNase), Tn5 transposase, DNase I, Endonuclease V, or Fragmentase, the mechanical method is, for example, ultrasonic fragmentation, centrifugation, or heat shock, and the chemical method is, for example, treating the nucleic acid sample with a chemical reagent to break it into multiple fragments.

[0026] In some embodiments, the end repair comprises blunting the ends of the fragmented nucleic acids so that the fragmented nucleic acids can be ligated to blunt-ended adapters.

[0027] In some embodiments, the end repair further comprises the step of adding A, that is, adding adenine deoxyribonucleotide to the 3' end after the end is filled, so as to connect the nucleic acid fragment to the linker through AT.

[0028] In some embodiments, the enzyme used to blunt-end the fragmented nucleic acid is, for example, T4 DNA polymerase, and the enzyme used in the A addition step is, for example, Klenow DNA polymerase (3'-5' exo-).

[0029] In some embodiments, the nucleic acid sample is double-stranded DNA, double-stranded RNA, DNA-RNA hybrid double-stranded, single-stranded DNA and / or single-stranded RNA. In some embodiments, when the sample is single-stranded DNA, the method further comprises the step of obtaining double-stranded DNA by random primers. In some embodiments, when the sample is single-stranded RNA, the method further comprises the step of reverse transcription.

[0030] In some embodiments, the purification step uses a magnetic bead method, a high salt precipitation method, a centrifugal column method, or a phenol-chloroform extraction method for purification; preferably, the magnetic bead method.

[0031] In a third aspect, the present application provides a method for constructing a library for studying protein-DNA interactions, the method comprising:

[0032] collecting a sample, wherein the sample is cells, permeabilized cells, chromosomes and / or chromatin;

[0033] Incubating the sample with a target protein binding antibody, wherein the target protein binding antibody binds to the target protein;

[0034] Adding a nuclease to fragment the target DNA to obtain DNA fragments, wherein the nuclease is connected to a binder capable of binding to the target protein-binding antibody, and the target DNA is located near the target protein;

[0035] Optionally, purifying the DNA fragment;

[0036] Perform end repair on purified or unpurified DNA fragments;

[0037] Using the method provided in the first aspect of the present application, connecting the DNA fragment to a linker;

[0038] Optionally, purifying the adapter ligation product;

[0039] Optionally, amplifying the adapter-ligated product or its purified product;

[0040] Optionally, the amplified product is purified.

[0041] In some embodiments, when ligated with the adaptor AT, it is necessary to add A to the DNA fragment.

[0042] In some embodiments, the cells are permeabilized or perforated, for example, by adding digitonin, polyethylene glycol octylphenyl ether, ethylphenyl polyethylene glycol or Tween to treat the cells, preferably digitonin. In some embodiments, the method comprises fixing the cells before or after permeabilization on magnetic beads, preferably concanavalin A magnetic beads.

[0043] In some embodiments, the nuclease is micrococcal nuclease MNase. In some embodiments, the conjugate of the target protein binding antibody is, for example, ProteinA, Protein G or ProteinAG, preferably ProteinAG. In some embodiments, the method further comprises adding an antibody (secondary antibody) to the target protein binding antibody for incubation, wherein the secondary antibody is used to increase the number of proteinAG binding regions so that the nuclease-antibody conjugate is more effectively bound to the target protein binding antibody.

[0044] In some embodiments, the method further comprises the step of activating the nuclease, such as by adding a divalent metal cation, such as Ca 2+ Mg 2+ , Mn 2+ 、Zn 2+ 、Co 2+ , or a mixture of the above ions, preferably with the addition of Ca 2+ .

[0045] In some embodiments, the target protein is a transcription factor or a histone.

[0046] In some embodiments, the target protein is a histone, such as at least one of H3k27me3, H3K4me3, H3K27ac, H3K4me1, H3K4me2, H2A, and / or Abf1.

[0047] In some embodiments, the target protein is a transcription factor, such as CTCF and / or Reb1.

[0048] In some embodiments, the cell is a human K562 cell.

[0049] In some embodiments, the number of cells is at least 100. In some embodiments, the number of cells is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000.

[0050] In some embodiments, the sample is from a eukaryotic organism or a prokaryotic organism, such as an animal, a plant, or a microorganism, such as a human, a mouse, a rat, Arabidopsis thaliana, or Saccharomyces cerevisiae.

[0051] In a fourth aspect, the present application provides a composition comprising isopropyl alcohol, guanidine thiocyanate, poloxamer, Tween 20 and SDS.

[0052] In some embodiments, the volume fraction of the isopropanol is 25%-95%, preferably 40%-90%, including, for example, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% within the range. In some embodiments, the concentration of the guanidine isothiocyanate is 10-30M, preferably 10-20M, including, for example, 10M, 11M, 12M, 13M, 14M, 15M, 16M, 17M, 18M, 19M or 20M within the range. In some embodiments, the concentration of the poloxamer is 1%-15%, preferably 1%-10%, including, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% within the range. In some embodiments, the concentration of Tween 20 is 0.1%-1%, preferably 0.1%-0.5%, including, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5% within the range. In some embodiments, the mass concentration of SDS is 0.1%-2%, preferably 0.2%-1%, including, for example, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95% or 1% within the range.

[0053] In some embodiments, the composition comprises 90% isopropanol, 15M guanidine thiocyanate, 4% poloxamer, 0.2% Tween 20, and 0.6% SDS; in some embodiments, the composition comprises 80% isopropanol, 15M guanidine thiocyanate, 4% poloxamer, 0.2% Tween 20, and 0.6% SDS; in some embodiments, the composition comprises 70% isopropanol, 15M guanidine thiocyanate, 4% poloxamer, 0.2% Tween 20, and 0.6% SDS; in some embodiments, the composition comprises 60% isopropanol, 15M guanidine thiocyanate, 4% poloxamer, 0.2% Tween 20, and 0.6% SDS; in some embodiments, the composition comprises 50% isopropanol, 15M guanidine thiocyanate, 4% poloxamer, 0.2% Tween 20 and 0.6% SDS; in some embodiments, the composition comprises 40% isopropanol, 15M guanidine thiocyanate, 4% poloxamer, 0.2% Tween 20 and 0.6% SDS.

[0054] In a fifth aspect, the present application provides use of the composition of any embodiment of the fourth aspect in nucleic acid purification.

[0055] In a sixth aspect, the present application provides the use of the composition of any embodiment of the fourth aspect in the nucleic acid purification step in the methods described in the second and third aspects; in particular, for purifying 150-300 bp nucleic acid fragments.

[0056] In a seventh aspect, the present application provides a kit comprising Sso7d-T4 DNA ligase and its reaction buffer, a nucleic acid fragmentation reagent and an end repair reagent, wherein the reaction buffer enables the Sso7d-T4 DNA ligase to exert its ligation activity.

[0057] In some embodiments, the nucleic acid fragmentation reagent is, for example, a fragmentation enzyme such as MNase and its reaction buffer.

[0058] In some embodiments, the end repair reagent is, for example, T4 DNA polymerase, Klenow DNA polymerase, T4 polynucleotide kinase, or a mixture of any two or three thereof, and a reaction buffer for the enzymes.

[0059] In some embodiments, the kit further comprises a nucleic acid purification reagent, such as a magnetic bead purification reagent. In some embodiments, the nucleic acid purification reagent comprises a purification buffer as described in multiple embodiments of the fourth aspect of the present application.

[0060] In some embodiments, the kit further comprises a connector; preferably, the connector is a blunt-end connector, a Y-type connector, or a bubble connector.

[0061] In some embodiments, the kit further comprises an amplification reagent such as a polymerase for amplification and a reaction buffer.

[0062] In some embodiments, the Sso7d-T4 DNA ligase comprises the amino acid sequence shown in SEQ ID NO.1.

[0063] In some embodiments, the kit is used to implement the method provided in the first aspect, the second aspect, the third aspect and / or the fifth aspect of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 :Schematic diagram of ChIP technology;

[0065] Figure 2 : Cut&Run technology schematic diagram;

[0066] Figure 3 :Schematic diagram of TF mainly binding to NDR region;

[0067] Figure 4A :The influence of different purification buffers on the purification results of fragmentation products; Figure 4B :The effect of different purification buffers on the proportion of 150-300bp small fragments in the purified product; Figure 4C :The effect of different purification buffers on the concentration and purity of the purified product;

[0068] Figure 5A :The influence of different purification buffers on the construction of small fragment recovery product library; Figure 5B :The effect of different purification buffers on the proportion of 150-300bp small fragments in the library products;

[0069] Fig. 6A : Comparison of library products obtained by two library construction methods; Figure 6B : The proportion of small fragments of 150-300 bp in the library products obtained by the two library construction methods;

[0070] Figure 7 : Comparison of Heatmaps of library sequencing results obtained by two library construction methods;

[0071] Fig. 8A : Heatmap of library sequencing results obtained by comparing two library construction methods with different cell input amounts using CTCF as the target; Figure 8B : Heatmap of library sequencing results obtained by comparing two library construction methods with different cell input amounts using H3k27me3 as the target;

[0072] Fig.9A : Comparison of the IGV signal diagram of the library sequencing results obtained by using CTCF as the target with different cell input amounts in two library construction methods; Fig. 9B : IGV signal diagram of library sequencing results obtained by comparing two library construction methods with different cell input amounts using H3k27me3 as the target.

[0073] Specific implementation (embodiment)

[0074] The technical solution of the present application is further explained below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are merely simple examples of the present application and do not represent or limit the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims.

[0075] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0076] Experimental reagents

[0077] Use pure water as solvent to prepare the following purification buffer:

[0078] Purification buffer 1: contains 15 M guanidine thiocyanate, 40% isopropanol, 4% poloxamer, 0.2% tween20, and 0.6% SDS;

[0079] Purification buffer 2: contains 15 M guanidine thiocyanate, 50% isopropanol, 4% poloxamer, 0.2% tween20, and 0.6% SDS;

[0080] Purification buffer 3: contains 15 M guanidine thiocyanate, 60% isopropanol, 4% poloxamer, 0.2% tween20, and 0.6% SDS;

[0081] Purification buffer 4: contains 15 M guanidine thiocyanate, 70% isopropanol, 4% poloxamer, 0.2% tween20, and 0.6% SDS;

[0082] Purification buffer 5: contains 15 M guanidine thiocyanate, 80% isopropanol, 4% poloxamer, 0.2% tween20, and 0.6% SDS;

[0083] Purification buffer 6: contains 15 M guanidine thiocyanate, 90% isopropanol, 4% poloxamer, 0.2% tween20, and 0.6% SDS;

[0084] Purification buffer 7: contains 15 M guanidine thiocyanate, 4% poloxamer, 0.2% Tween 20, and 0.6% SDS.

[0085] Example 1

[0086] Taking the transcription factor CTCF of K562 cells (Wuhan Pronocell Life Science Co., Ltd., Catalog No. CL-0130) as the target, according to the method steps in the literature “Targeted in situ genome-wide profiling with high efficiency for low cell numbers”, 7 K562 cells (each cell amount is 10000 cells) were subjected to Cut&Run experiment to fragment the cell chromatin. The CTCF target antibody used was Active motif product (Cat.No.61932), and the PAG-MNase enzyme was Cell Signaling Technology product (Cat.No.40366S). Finally, 7 fragmented products were obtained, mixed, and then divided into 7 equal parts, each with a volume of 200 μl, to ensure that the amount of each product was the same.

[0087] Example 2

[0088] The fragmented cell product in Example 1 was subjected to magnetic bead DNA purification, and different purification buffers were used to illustrate the effect of isopropanol on the purification of small fragments. The specific operation was as follows:

[0089] 5 μl Proteinase K and 40 μl VAHTSParticles G (Nanjing Novozyme, catalog number: N903) were added to the 7 samples obtained in Example 1, and then 300 μl purification buffer 1-7 was added to the 7 samples, vortexed and mixed, and allowed to stand for 10 min, during which time the mixture was mixed by inverting 2-3 times;

[0090] After a brief centrifugation, place on a magnetic stand and let stand for 30s-2min, then discard the supernatant;

[0091] Add 900 μl buffer WA (NanoVizan, catalog number: N903), vortex for 15 seconds, centrifuge briefly, place on a magnetic rack, let stand for 30 seconds to 2 minutes, and discard the supernatant;

[0092] Add 900 μl buffer WB (Nanjing Novozyme, catalog number: N903), vortex for 15 seconds, centrifuge briefly, place on a magnetic rack, let stand for 30 seconds to 2 minutes, discard the supernatant, and repeat this step once;

[0093] Dry at room temperature for 5-20 minutes until the surface of the magnetic beads is no longer reflective;

[0094] Each purified sample was eluted with 22 μl of ddH2O, allowed to stand for 5 min, and placed on a magnetic rack after brief centrifugation for 30 s-2 min;

[0095] The 7 purified samples were tested by Agilent 2100 Bioanalyzer to detect the peak shape of the purified nucleic acid and to detect the 260 / 280 and 260 / 230 performances.

[0096] Results Analysis

[0097] like Figure 4A -C, where Figure 4A The "normal buffer" in the table refers to the purification buffer containing 0% isopropanol (i.e., purification buffer 7). Figure 4CThe "optimized buffer" refers to a purification buffer containing 90% isopropanol (i.e., purification buffer 6). By adding isopropanol to the purification buffer, the proportion of 150bp-300bp small fragments in the total recovered products can be increased. Different concentrations of isopropanol have different purification and recovery efficiencies for small fragments. The concentration, 260 / 280 and 260 / 230 performance of the purified products treated with purification buffers 1-6 are significantly better than those of the products purified by purification buffer 7. Among them, purification buffer 6, i.e., a purification buffer with a concentration of 90% isopropanol, is best for improving the purification yield of small fragment DNA.

[0098] Example 3

[0099] Taking K562 cell transcription factor CTCF as the target, Cut&Run chromatin fragmentation was performed according to Example 1, and the fragmentation products were purified by purification buffer 6 and purification buffer 7, and the differences in the library construction direction between the two were compared. The specific operations were as follows:

[0100] According to the method of Example 1, two portions of 10,000 K562 cells were subjected to Cut&Run chromatin fragmentation. After mixing, the fragmentation products were equally divided into two portions. Each portion was purified using purification buffer 6 and purification buffer 7 according to the method steps of Example 2, and 20 μl of each product was taken for library construction.

[0101] The library of the above two purified fragment products was constructed using Nanjing Novozyme ND608 product, and the purified products were end-filled and damaged according to the following system:

[0102] Components volume InputDNA 20μl EndPrepEnzyme 3μl EndPrepBuffer 7μl <![CDATA[ddH2O]]> 30μl Total volume 60μl

[0103] After mixing the above components, perform the following final repair procedures:

[0104] temperature time 105℃ hot cover on 20℃ 30min 65℃ 30min 4℃ Hold

[0105] The above products were connected to the adapters. The DNA Adapter X adapters were from Nanjing Novozymes N321 / N322. The adapters were diluted 1:100 and used. The following system was prepared for the connection:

[0106] Components volume Unfinished product 60μl RapidLigationbuffer2 30μl RapidDNALigase 10μl DNAAdapterX 5μl <![CDATA[ddH2O]]> 5μl Total volume 110μl

[0107] After mixing the above components, put them on the machine and carry out the following procedures:

[0108] temperature time 105℃ hot cover on 20℃ 15min 4℃ Hold

[0109] 165 μl of DNA magnetic beads (Nanjing Novozyme, catalog number N411) were used to purify the product after the connector connection, 22 μl of pure water was used for elution, and 20 μl was taken for library amplification.

[0110] Prepare the library amplification components according to the following system:

[0111] Components volume Purification of the product after ligation 20μl PCR Primer Mix 3 for Illmina 5μl VAHTSHiFiAmplificationMix 25μl Total volume 50μl

[0112] After mixing the above components, perform the following amplification procedure on the machine:

[0113]

[0114] 100 μl of DNA magnetic beads (Nanjing Novozyme, catalog number N411) were used to purify the amplified product, 22 μl of pure water was used for elution, the purified product was tested for peak shape using Agilent 2100 Bioanalyzer, and the two libraries were sequenced.

[0115] Results Analysis

[0116] like Figure 5A -B, the proportion of 150-300bp small fragment library in the library product constructed by purification buffer 6 (optimized buffer) containing 90% isopropanol is higher than the proportion of small fragments in the library product constructed by purification buffer 7 (ordinary buffer), indicating that isopropanol not only directly improves the recovery rate of 150-300bp small fragments after fragmentation, but also ultimately increases the proportion of 150-300bp small fragment library in the library product.

[0117] Example 4

[0118] Using transcription factor CTCF as the target protein, K562 cells were subjected to Cut&Run chromatin fragmentation, and the fragmentation products were purified by purification buffer 6. When the purified products were connected with adapters, conventional T4 DNA ligase and fusion Sso7d-T4 DNA ligase were used respectively to compare the effects of the two ligases on library construction and sequencing. The Sso7d-T4 DNA ligase fusion protein was prepared according to the reference (Wilson RH, Morton SK, Deiderick H, Gerth ML, Paul HA, Gerber I, Patel A, Ellington AD, Hunicke-Smith SP, Patrick WM. Engineered DNA ligases with improved activities in vitro. Protein Eng Des Sel. 2013 Jul; 26 (7): 471-8.).

[0119] According to the method of Example 1, two portions of 100 cells and two portions of 10,000 cells were respectively subjected to Cut&Run chromatin fragmentation. The fragmentation products were purified using purification buffer 6 according to the method steps of Example 2. Each purified product with different input amounts was mixed and then divided equally into two portions, each with 20 μl.

[0120] The library was constructed according to the method and steps of Example 3. During the library construction process, two fragmented purified products with the same input amount were connected by connectors using 2 μl 0.2M T4 DNA ligase (Vazyme N103) and 2 μl 0.2M fusion Sso7d-T4 DNA ligase, respectively. The amplified and purified library was subjected to Agilent 2100 Bioanalyzer library peak detection and sequencing.

[0121] Results Analysis

[0122] like Fig. 6A -B is the library output result of 10,000 cells input, compared with the conventional T4 DNA ligase ( Fig. 6A -B) and the resulting library product was ligated with a fusion Sso7d-T4 DNA ligase (i.e. Fig. 6A -B) by linker ligation, and the library product constructed by Sso7d-T4 DNA ligase contained 150-300 bp small fragments accounting for up to 53%.

[0123] like Figure 7 ， The Sso7d-T4 DNA ligase group improved the performance of the Cut&Run technology. The higher the target signal value, the better the enrichment and the higher the signal-to-noise ratio. Through Sso7d-T4 DNA ligase, the CTCF signal value of the 10,000 cell input group increased from 80 to 175, and the CTCF signal value of the 100 cell input group increased from 14 to 60.

[0124] Example 5

[0125] Taking transcription factor CTCF protein and histone H3k27me3 as target proteins (using Cat. No. 9733 product of Cell Signaling Technology as target antibody), each target protein was subjected to Cut&Run chromatin fragmentation for 2 samples of 10,000 cells and 2 samples of 100 cells according to the method of Examples 1-3. Each two samples of the same cell amount were mixed and divided into 2 equal parts after mixing. Each part was respectively constructed using purification buffer 6 in combination with fusion Sso7d-T4 DNA ligase to construct a library, and purification buffer 7 in combination with conventional T4 DNA ligase to construct a library. The effect of optimizing the combination of purification buffer and fusion Sso7d-T4 DNA ligase on library construction and sequencing results was studied.

[0126] Results Analysis

[0127] like Fig. 8A -B, where "normal buffer" refers to purification buffer 7, and "optimized buffer" refers to purification buffer 6. The heatmap shows that purification buffer 6 and fusion Sso7d-T4 DNA ligase (i.e. Fig. 8A -B) combined with Sso-T4 DNA Ligase increased the signal value of CTCF and H3K27me3 targets. The higher the signal value, the better the enrichment and the higher the signal-to-noise ratio, indicating that compared with purification buffer 7 and conventional T4 DNA ligase (i.e. Fig. 8A -B) in combination with purification buffer 6 and fusion Sso7d-T4 DNA ligase, the quality of Cut&Run sequencing was improved, and good performance was also shown at low cell input (100 cells input).

[0128] like Fig.9A -B, where "normal buffer" refers to purification buffer 7, and "optimized buffer" refers to purification buffer 6. The IGV signal graph shows that purification buffer 6 and fusion Sso7d-T4 DNA ligase (i.e. Fig.9A -B) combined with Sso-T4 DNALigase improved the quality of Cut&Run sequencing of CTCF and H3k27me3 targets, and also showed good performance at low cell starting amount (100 cells input).

Claims

1. A composition, characterized in that The composition consists of isopropyl alcohol, guanidine thiocyanate, poloxamer, Tween 20 and SDS, wherein: The volume fraction of the isopropanol is 70%-90%; The concentration of the guanidine isothiocyanate is 15M; The concentration of the poloxamer is 4%; The concentration of Tween 20 is 0.2%; The mass concentration of the SDS is 0.6%.

2. The composition according to claim 1, wherein The composition contains 15M guanidine thiocyanate, 4% poloxamer, 0.2% Tween 20, 0.6% SDS and 70% isopropanol; or, the composition contains 15M guanidine thiocyanate, 4% poloxamer, 0.2% Tween 20, 0.6% SDS and 80% isopropanol; or, the composition contains 15M guanidine thiocyanate, 4% poloxamer, 0.2% Tween 20, 0.6% SDS and 90% isopropanol.

3. Use of the composition according to any one of claims 1 to 2 in nucleic acid purification.

4. A method for constructing a nucleic acid library, characterized in that: The method comprises: Collecting a nucleic acid sample, and shearing the nucleic acid sample to obtain fragmented nucleic acid; Purification of fragmented nucleic acids; Performing end repair on the fragmented nucleic acid to obtain nucleic acid fragments; Using a ligase to connect the nucleic acid fragments and the adapter; Optionally, amplifying the ligation product; Wherein, the fragmented nucleic acid is purified using the composition described in any one of claims 1 to 2.

5. A method for constructing a library for studying protein-DNA interactions, characterized in that: The method comprises: collecting a sample, wherein the sample is cells, permeabilized cells, chromosomes and / or chromatin; Incubating the sample with a target protein binding antibody, wherein the target protein binding antibody binds to the target protein; Adding a nuclease to fragment the target DNA to obtain DNA fragments, wherein the nuclease is connected to a binder capable of binding to the target protein-binding antibody, and the target DNA is located near the target protein; purifying the DNA fragment; Perform end repair on the purified DNA fragments; Use a ligase to connect the DNA fragment to the adapter; optionally, amplify the adapter ligation product, Wherein, the DNA fragment is purified using the composition described in any one of claims 1-2.

6. A nucleic acid purification reagent, characterized in that: A composition comprising any one of claims 1 to 2.

7. A kit, characterized in that: Contains the nucleic acid purification reagent according to claim 6.

8. The kit according to claim 7, further comprising a ligase and a reaction buffer thereof, a nucleic acid fragmentation reagent, an end repair reagent, and at least one of a linker and an amplification reagent.

9. The kit according to claim 8, wherein the ligase comprises an amino acid sequence as shown in SEQ ID NO: 1; the nucleic acid fragmentation reagent is a fragmentation enzyme MNase and a reaction buffer thereof; the end repair reagent is T4 DNA polymerase, Klenow DNA polymerase, T4 polynucleotide kinase or a mixture of any two or three thereof, and a reaction buffer of the enzyme; the connector is a blunt-end connector, a Y-shaped connector or a bubble connector; and the amplification reagent is an amplification polymerase and a reaction buffer.

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