CN112359092B - Construction method of genome short fragment library - Google Patents
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Abstract
The invention relates to the field of bioengineering, in particular to a construction method of a genome short fragment library. The invention comprises the following steps: s1, extracting whole genome of a cell; s2, genome fragmentation and product recovery and purification; s3, mnase enzyme digestion and product recovery; s4, electrophoresis and glue recovery; s5, terminal repair and joint connection; s6, cloning a vector and constructing a library. The short fragment DNA library with the length of 15-50bp, which is enriched by the non-sequence preference enzyme digestion method, covers the complete information of the whole genome of the animal, and the method has the advantages of simple operation and stable technical system, and provides convenience for sequence information analysis of regulatory element motifs.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a construction method of a genome short fragment library.
Background
In the field of bioengineering, model organisms (common laboratory animals, plants or microbial objects, such as mice, drosophila, arabidopsis thaliana, etc.) have accurate reference genomes and complete functional element annotation information, which can provide reliable basic data for experiments. However, existing methods of genome fragmentation have difficulty achieving enrichment of small fragments. The conventional ultrasonic breaking method has a limited physical shearing force generated by ultrasonic waves when DNA fragmentation is performed, and is difficult to break DNA molecules to below 100 bp. Commercial DNA fragment enzymes used for breaking DNA by an enzyme digestion method have certain enzyme digestion site preference, and have limited enzyme digestion activity, so that DNA fragments below 50bp are difficult to enrich.
Disclosure of Invention
The invention aims at providing a method for efficiently enriching a mouse genome short fragment library.
The length distribution range of the short fragments of the genome, which is aimed at by the invention, is 15-50bp, and the short fragment library is constructed mainly for meeting the research requirement of functional genomics, namely, the transcriptional activation of the short fragment DNA motif as an independent regulatory element is analyzed through analyzing the sequence characteristics and functions of the short fragment library which covers the whole genome sequence information.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the construction method of the genome short fragment library comprises the following steps:
s1, extracting whole genome of a cell;
s2, genome fragmentation and product recovery and purification: breaking the whole genome extracted in the step S1 and recovering a broken product;
s3, mnase digestion and product recovery: carrying out Mnase digestion on the broken product in the step S2 and recovering digested products;
s4, electrophoresis and glue recovery: electrophoresis is carried out on the enzyme digestion product obtained in the step S3, and fragments with the range of 15-50bp are cut and recovered;
s5, terminal repair and joint connection: performing end repair and A addition on the DNA fragment recovered in the step S4, and performing adapter ligation on the DNA fragment subjected to end repair to obtain an adapter ligation product;
s6, vector cloning and library construction: adding a carrier homology arm into the adapter connection product obtained in the step S5, then carrying out carrier cloning to obtain a recombinant product, carrying out transformation culture on the recombinant product, extracting recombinant plasmid, carrying out amplification by taking the recombinant plasmid as a template, and constructing a DNA sequencing library by taking the amplified product as input DNA.
The method for constructing a genome short fragment library according to the present invention, wherein preferably, the step S1 comprises the steps of:
s11, digestion and lysis of cultured cells;
s12, purifying by column: purifying the digested and cracked digestive juice by using an adsorption column to obtain purified genome DNA.
The method for constructing a genome short fragment library according to the present invention, wherein preferably, the step S2 comprises the steps of:
s21, breaking the whole genome DNA of the cells by using an ultrasonic instrument;
s22, purifying the broken DNA fragments by using an adsorption column to obtain purified broken products.
According to the method for constructing a genomic short fragment library of the present invention, preferably, step S4 is performed by TBE PAGE.
The method for constructing a genome short fragment library according to the present invention, wherein preferably, the step S5 comprises the steps of:
s51, repairing the tail end: performing END repair on the DNA fragment recovered in the step S4 by using an END prepmix, and adding A;
s52, joint connection: performing adapter ligation on the DNA fragments with the repaired tail ends by using a DNA adapter to obtain an adapter ligation product;
s53, magnetic bead purification of a joint connection product: and (3) performing magnetic bead purification on the adapter ligation product to obtain a purified adapter ligation product.
The method for constructing a genome short fragment library according to the present invention, wherein preferably, the step S6 comprises the steps of:
s61, adding a carrier homology arm: adding a carrier homology arm to the adapter ligation product by a PCR reaction;
s62, recovering PCR products: cutting a target band in a range of 110-150bp, and recovering and purifying;
s63, cloning a vector: connecting and amplifying the adapter connecting product with a carrier to obtain a recombinant product;
s64, transformation culture: allowing the recombinant product to enter competent cells to complete transformation and culturing the transformed cells;
s65, plasmid extraction: extracting recombinant plasmid from the cultured transformed cells;
s66, library construction: amplifying a target sequence containing an insert fragment by taking the recombinant plasmid as a template, recovering and purifying a product with the size of 209-244bp, and constructing a DNA sequencing library by taking the expanded and recovered product as input DNA, wherein the size of a library fragment is 332-367bp.
The construction method of the genome short fragment library has wide applicability and can be used for constructing genome short fragment libraries derived from animal and plant, microbial cells or tissues. Is particularly suitable for animals, such as but not limited to laboratory animals like mice, drosophila, zebra fish, etc.
In order to obtain a short-fragment DNA library, the invention combines a physical method and an enzyme digestion method, and optimizes the enzyme digestion condition to achieve the purpose of short-fragment enrichment by selecting an endonuclease MNase with stronger enzyme digestion activity and low preference. Meanwhile, the digestion product of MNase is a blunt-ended DNA molecule, which provides convenience for subsequent library construction. In addition, the invention adopts the mode of adding adapter and homologous recombination in the carrier construction, and provides a simple, convenient and efficient carrier construction method.
Compared with the prior art, the invention has the advantages and positive effects that: the short fragment DNA library with the length of 15-50bp, which is enriched by the enzyme digestion method with disorder preference, covers the complete information of the whole genome of the organism, has simple and convenient operation and stable technical system, and provides convenience for sequence information analysis of regulatory element motifs.
Drawings
FIG. 1 shows the recovery of a PAGE gel of MNAse cleavage products according to an embodiment of the invention;
FIG. 2 shows the recovery of a PAGE gel of PCR amplification products linked to homology arms according to an embodiment of the present invention;
FIG. 3 is a schematic illustration of segment lengths according to an embodiment of the present invention;
FIG. 4A is a schematic diagram of a digestion system for constructing a plasmid library according to an embodiment of the present invention;
FIG. 4B shows the recovery of a PAGE gel of the enzyme-cleaved products constructed from the plasmid library in the examples of the present invention;
FIG. 4C is a photograph of a recombinant plasmid PAGE gel constructed from a plasmid library according to an embodiment of the present invention;
FIG. 5 shows the amplification of target fragments of a plasmid library according to an embodiment of the present invention;
FIG. 6A is a fragment of interest constructed from a plasmid library according to an embodiment of the present invention;
FIG. 6B is a graph showing the results of library quality control using 2100 constructed plasmid library according to the example of the present invention.
Detailed Description
The following describes the technical scheme of the present invention in detail by referring to examples. The reagents and biological materials used hereinafter are commercial products unless otherwise specified. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The experimental methods of molecular biology, which are not specifically described in the examples, are all carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (fourth edition) j.
The invention provides a construction method of a genome short fragment library, which specifically comprises the following steps:
s1, extracting whole genome of a cell;
s2, ultrasonic fragmentation of genome and recovery and purification of products;
s3, mnase enzyme digestion and product recovery;
s4, TBE PAGE electrophoresis and glue recovery;
s5, terminal repair and joint connection;
s6, cloning a vector and controlling the quality of a library;
further, in the step S1, the extraction of the genome of the Cell is performed by using FastPure Cell/Tissue DNA Isolation Mini Kit (DC 102, vazyme).
S11, digestion and lysis of cultured cells
(1) Total cell count 1X 10 6 -5×10 6 And each. 4Cells were collected by centrifugation at 00 and x g for 5min and the supernatant was discarded. 220uL PBS, 10uL RNase Solution and 20uL PK working fluid were added to the samples, and the cells were resuspended. Standing at room temperature for 15min.
(2) 250uL Buffer GB was added to the cell resuspension and vortexed. Water bath at 65 ℃ for 15min.
S12, purifying by column
(3) 250uL absolute ethanol is added into the digestion liquid, and the mixture is stirred and mixed for 15-20sec.
(4) The gDNA Columns were placed in Collection Tubes 2 mL. Transferring the mixed solution (including sediment) obtained in the last step into an adsorption column. Centrifuging for 1min at 10,000-12,000Xg.
(5) The filtrate was discarded and the column was placed in a collection tube. 500uL Washing Buffer A was added to the column. Centrifuging for 1min at 10,000-12,000Xg.
(6) The filtrate was discarded and the column was placed in a collection tube. 650uL Washing Buffer B was added to the adsorption column. Centrifuging for 1min at 10,000-12,000Xg.
(7) And (4) repeating the step 4.
(8) The filtrate was discarded and the column was placed in a collection tube. Centrifuge for 2min with 10,000-12,000Xg empty tube.
(9) The column was placed in a new 1.5mL centrifuge tube. 100uL of Elutionion Buffer preheated to 70 ℃ is added to the center of the membrane of the adsorption column and left at room temperature for 3min. Centrifuging for 1min at 10,000-12,000Xg.
The adsorption column is removed, the DNA is stored at 2-8 ℃ and the long-term storage is required to be placed at-20 ℃.
Further, the step S2 includes the following steps:
s21, taking 1-3ug of genome DNA to 300uL of TE buffer, and carrying out DNA disruption by using a biomaker ultrasonic instrument.
S22, purifying by a column.
(1) Adding 5 times of volume DNA binding buffer, shaking and mixing
(2) The mixture was transferred to a Zymo-Spin Column,30s centrifuge, 10,000-12,000Xg, room temperature.
(3) 200uL DNA wash buffer for 2 times, 30s centrifugation, 10,000-12,000Xg, room temperature.
(4) 25uL DNA Elution buffer was added to the column, incubated for 1min at room temperature, centrifuged for 30s, 10,000-12,000Xg at room temperature.
Further, the step S3 includes the following steps:
s31, taking 1-3ug DNA ultrasonic disruption product (100-200 bp), and carrying out Mnase digestion (4 ℃,30min,100uL system) with the working concentration of Mnase of 1U/uL.
S32, recycling enzyme cutting products.
(1) Add to 100uL Oligo Binding Buffer to 50ul of sample.
(2) Adding 400uL of 95-100% ethanol, mixing with a pipette, and transferring to Zymo-Spin in a collecting tube TM In Column.
(3) More than or equal to 10,000Xg, centrifuging for 30s, and discarding the waste liquid.
(4) Adding 750uL of DNA washing buffer solution into the column, centrifuging for 30s at a speed of more than or equal to 10,000Xg, and discarding the waste liquid; the maximum rotation speed is centrifuged for 1min.
(5) The column was placed in a fresh centrifuge tube, directly added with 15-50uL of water or TE onto the membrane, and centrifuged at 10,000Xg for 30s for elution.
Further, the step S4 includes the following steps:
s41, TBE PAGE electrophoresis.
(1) And (3) glue preparation: 12% PAGE:1.5mm plate, 15mL gel, and the remaining additive ingredients are shown in Table 1 below.
TABLE 1 internal gum ingredients
(2) Electrophoresis, 0.5 XTBE electrophoresis solution, parameters: constant pressure 60V,15min;120V,40min.
(3) gel staining, SYBR Gold (10000X) diluted with 1 XTBE, staining for 5min. The cleavage is in the range of 15-50 bp.
S42, punching 3-4 holes on the bottom of a centrifugal tube of 0.5mL DNase-free by using a 21G needle.
S43, placing the punched 0.5mL centrifuge tube into a 1.5mL DNase-free centrifuge tube, and placing the gel recovery into the 0.5mL centrifuge tube.
S44, 20,000Xg ionThe heart was left for 4min, gel fragments were collected in a 1.5mL centrifuge tube, and 200uLRNase-free H was added 2 Mixing evenly. Incubating at 70 ℃ for 10min; the mixture was vortexed for 30s.
S45, transferring the sol to microcentrifuge tube filter by using a 1000uL tip with the tip cut off.
S46, 20,000Xg, at room temperature, for 3min. 2-3 reaction samples were pooled, with a volume of about 400-600uL.
S47, adding 25uL,5M NaCl,2uL,15mg/mL GlycoBlue and 750uL isopropanol, uniformly mixing, and precipitating at-20 ℃ for more than or equal to 1h or overnight.
S48, 20,000Xg centrifugation for 30min,4℃and supernatant removal. 750uL 80% glacial ethanol was washed once. 20,000Xg was centrifuged at 2min at 4℃and the supernatant was removed.
S49, air drying at room temperature for 10min.15uL ddH 2 O dissolves DNA precipitate and is preserved for a long period of time at-20 ℃.
Further, in the step S5:
s51, repairing the tail end:
the reaction system was configured on ice: DNA small fragment product, 15-50uL,END prep mix4, 15uL,ddH 2 O: supplementing to 65uL, lightly blowing 10 times by a pipette, uniformly mixing, performing instantaneous centrifugation for 3-5s, and performing PCR reaction under the reaction conditions: the heat cover was temporarily stored at 105 ℃,20 ℃,15min,65 ℃,15min,4 ℃.
S52, joint connection:
the reaction system was configured on ice: the end repair products, 65uL,rapid ligation buffer2, 25uL,rapid DNAligase,5uL,DNA adapter x,5uL, were gently mixed by pipetting 10 down and centrifuged for 3-5s transiently for PCR reaction under the following conditions: the heat cover was temporarily stored at 105℃at 20℃for 15min at 4 ℃.
S53, magnetic bead purification of a joint connection product:
VASHTS DNA clear Beads equilibrate to room temperature in advance for 30min, shake 5s uniformly mixing the Beads with a front vortex, absorb 100uL Beads to 100uL joint connection products (volume ratio 1:1), shake 5s uniformly mixing, incubate for 5min at room temperature, instantly centrifuge for 3-5s, place the magnetic Beads and liquid on a magnetic rack, stand for 5min, carefully absorb supernatant after the solution is clarified, rinse the Beads twice with 200uL freshly prepared 80% ethanol, incubate for 30s at room temperature, carefully absorb supernatant, uncover air dry the Beads for 5min until no ethanol remains, take the PCR tube out of the magnetic rack, perform elution: adding 25uL of eluent (10 mM Tris-HCl, pH 8.0-8.5), shaking with vortex for 5s, mixing, incubating at room temperature for 2min, centrifuging for 3-5s instantaneously, separating magnetic beads and liquid with a magnetic rack, standing for 5min, carefully sucking 23uL of supernatant into a new EP tube after the solution is clarified, and preserving at-20deg.C for a long time.
Further, in the step S6:
s61, adding a carrier homology arm:
the vector homology arms were added by PCR reaction. PCR reaction 25uL: KAPA HIFI Hotstart Mix,12.5uL, adapter-F primer, 0.5uL, adapter-R primer, 0.5uL, adapter ligation product, 11.5uL.
S62, recycling a PCR product PAGE gel:
all 25uL PCR products are loaded into spot sample holes, electrophoresis is carried out for 30min under 120V voltage, and target bands in the range of 110-150bp are cut for recovery and purification. The experimental procedure is detailed in S41.
S63, cloning a vector: kit Clone Express II one step cloning kit, vazyme, C112
The optimal dosage is as follows: carrier amount (0.03 pmol) = (0.02 x carrier base number) ng
Amount of insert (0.06 pmol) = (0.04 x number of fragment bases) ng
Gently beating for 10 times, mixing, and instantly centrifuging for 3-5 times. Incubation was carried out in a PCR apparatus at 37℃for 30min. Temporary storage is carried out at 4 ℃.
S64, transformation and fungus culture:
thawing competent cells on ice, adding 10uL of recombinant product into 100uL of competent cells, performing heat shock in a water bath at 42 ℃ for 45s, immediately cooling on ice for 2-3min, adding 900uL of LB (without antibiotics), resuscitating at 37 ℃ for 1h (200-250 rpm) in a constant temperature shaking table, adding 30mL of LB into each tube, 60uL of Amp, and shaking at 37 ℃ for 12-16h in a constant temperature shaking table at 200 rpm.
S65, plasmid extraction:
the bacterial liquid is centrifuged for 10min at the room temperature of 5000 Xg, bacterial precipitate is collected, supernatant is carefully removed, 250uL of Solution I/RNase A is added, suspension bacterial liquid is blown up and down by vortex or a pipetting gun, 250uL of Solution II is added, and the mixture is gently inverted and evenly mixed for 4-6 times, thus obtaining clear lysate. Incubation was performed at room temperature for 2min, 125uL of pre-chilled BufferN3 (placed on ice in advance), mixing was reversed several times until white flocculent precipitate appeared, centrifuging at 12000×g 25 ℃ for 10min, carefully transferring the supernatant to a clean 1.5mL centrifuge tube, and adding 0.1 volumes of ETR Solution. Mixing for 7-10 times, and incubating on ice for 10min, wherein the incubation process can be reversed several times. Incubation was carried out at 42℃for 5min, and the lysate was again clouded and centrifuged at 12000 Xg at 25℃for 3min. Transferring the supernatant to a new 1.5mL centrifuge tube, adding 0.5 times of 96% -100% ethanol (room temperature), mixing for 6-7 times, incubating for 1-2min at room temperature, collecting the mixed solution, adding into a clean HiBindTM DNA Mini column assembly and a 2mL collecting tube, centrifuging at 10000×g at room temperature for 1min, passing through the column, and discarding the filtrate. The column was washed with 500uL Buffer HB and centrifuged as above. The column was washed twice with 700 a uL DNAWash Buffer and centrifuged as above. The filtrate was discarded, the empty column was returned to the centrifuge tube, centrifuged at maximum speed (. Gtoreq.13000 Xg) for 2min, and the column was dried. The column was placed in a new 1.5mL centrifuge tube, 80uLEndotoxin-Free Elution Buffer was applied to the column and centrifuged at maximum speed (. Gtoreq.13000 Xg) for 1min to elute the plasmid.
S66, library preparation:
amplifying target sequences containing inserts by using the recombinant plasmid library as a template, and amplifying primer information: pMX-TY1, TGCAGGTGCCAGAACATTTC, pMX-TY2, GTGGCTTTACCAACAGTACC. And (3) recovering and purifying products with the size of 209-244bp by using a PAGE gel, wherein the gel recovery flow is shown as S62, constructing a DNA sequencing library by taking the PCR recovery product as input DNA, recovering and purifying target fragments by using agarose gel, wherein the library fragment size is 332-367bp. The library construction procedure is shown in S41.
Example 1
S1, whole genome extraction, and extraction of genome of mouse embryonic stem cells (mESCs, E14) is performed by using FastPure Cell/Tissue DNA Isolation Mini Kit (DC 102, vazyme):
s11, digestion and lysis of cultured cells
(1) The total cell amount cannot exceed 5×10 6 And each. Cells were collected by centrifugation at 400 and x g for 5min and the supernatant was discarded. 220uL PBS, 10uL RNase Solution and 20uL PK working fluid were added to the samples, and the cells were resuspended. Standing at room temperature for 15min.
(2) 250uL Buffer GB was added to the cell resuspension and vortexed. Water bath at 65 ℃ for 15min.
S12, purifying by column
(3) 250uL absolute ethanol is added into the digestion liquid, and the mixture is stirred and mixed for 15-20sec.
(4) The gDNA Columns were placed in Collection Tubes 2 mL. Transferring the mixed solution (including sediment) obtained in the last step into an adsorption column. Centrifuge at 12,000Xg for 1min.
(5) The filtrate was discarded and the column was placed in a collection tube. 500uL Washing Buffer A was added to the column. Centrifuge at 12,000Xg for 1min.
(6) The filtrate was discarded and the column was placed in a collection tube. 650uL Washing Buffer B was added to the adsorption column. Centrifuge at 12,000Xg for 1min.
(7) And (4) repeating the step 4.
(8) The filtrate was discarded and the column was placed in a collection tube. The tube was centrifuged at 12,000Xg for 2min.
(9) The column was placed in a new 1.5mL centrifuge tube. 100uL of Elutionion Buffer preheated to 70 ℃ is added to the center of the membrane of the adsorption column and left at room temperature for 3min. Centrifuge at 12,000Xg for 1min.
The adsorption column is removed, the DNA is stored at 2-8 ℃ and the long-term storage is required to be placed at-20 ℃.
S2, ultrasonic fragmentation of genome and recovery and purification of products:
s21, taking 3ug of genome DNA to 300uL of TE buffer, and carrying out DNA disruption by using a biomap ultrasonic instrument.
S22, purifying by a column.
(1) Adding 5 times of volume DNA binding buffer, shaking and mixing
(2) The mixture was transferred to a Zymo-Spin Column, centrifuged for 30s, 10000g, room temperature.
(3) 200uL DNA wash buffer for 2 times, 30s for centrifugation, 10000g, room temperature.
(4) 25uL DNA Elution buffer to the column, incubated at room temperature for 1min, centrifuged for 30s, 10000g, room temperature.
S3, mnase digestion and product recovery:
s31, taking 1ug of DNA ultrasonic disruption product (100-200 bp), and carrying out Mnase digestion (4 ℃,30min,100uL system), wherein the working concentration of Mnase is 1U/uL.
S32, recycling enzyme cutting products.
(1) Add to 100uL Oligo Binding Buffer to 50ul of sample.
(2) Adding 400uL of 95-100% ethanol, mixing with a pipette, and transferring to Zymo-Spin in a collecting tube TM In Column.
(3) More than or equal to 10,000Xg, centrifuging for 30s, and discarding the waste liquid.
(4) Adding 750uL of DNA washing buffer solution into the column, centrifuging for 30s at a speed of more than or equal to 10,000Xg, and discarding the waste liquid; the maximum rotation speed is centrifuged for 1min.
(5) The column was placed in a fresh centrifuge tube, directly inoculated with 15uL of water or TE onto the membrane, and centrifuged at 10,000Xg for 30s for elution.
S4, TBE PAGE electrophoresis and glue recovery:
s41, TBE PAGE electrophoresis.
(1) And (3) glue preparation: 12% PAGE:1.5mm plate, 15mL gel, the remaining additive ingredients are as follows in Table 2.
TABLE 2 internal gum ingredients of example 1
(2) Electrophoresis, 0.5 XTBE electrophoresis solution, parameters: constant pressure 60V,15min;120V,40min.
(3) gel staining, SYBR Gold (10000X) diluted with 1 XTBE, staining for 5min. The cleavage is in the range of 15-50 bp.
S42, punching 3-4 holes on the bottom of a centrifugal tube of 0.5mL DNase-free by using a 21G needle.
S43, placing the punched 0.5mL centrifuge tube into a 1.5mL DNase-free centrifuge tube, and placing the gel recovery into the 0.5mL centrifuge tube.
S44, centrifugation at 20,000Xg for 4min, gel fragments were collected in a 1.5mL centrifuge tube, and 200uLRNase-fre was addede H 2 Mixing evenly. Incubating at 70 ℃ for 10min; the mixture was vortexed for 30s.
S45, transferring the sol to microcentrifuge tube filter by using a 1000uL tip with the tip cut off.
S46, 20,000Xg, at room temperature, for 3min. 2-3 reaction samples were pooled, with a volume of about 400-600uL.
S47, adding 25uL,5M NaCl,2uL,15mg/mL GlycoBlue and 750uL isopropanol, uniformly mixing, and precipitating at-20 ℃ for more than or equal to 1h or overnight.
S48, 20,000Xg centrifugation for 30min,4℃and supernatant removal. 750uL 80% glacial ethanol was washed once. 20,000Xg was centrifuged at 2min at 4℃and the supernatant was removed.
S49, air drying at room temperature for 10min.15uL ddH 2 O dissolves DNA precipitate and is preserved for a long period of time at-20 ℃.
S5, terminal repair and joint connection:
s51, repairing the tail end:
the reaction system was configured on ice: DNA small fragment product, 30uL,END prep mix4, 15uL,ddH 2 O: supplementing to 65uL, lightly blowing 10 times by a pipette, uniformly mixing, performing instantaneous centrifugation for 3-5s, and performing PCR reaction under the reaction conditions: the heat cover was temporarily stored at 105 ℃,20 ℃,15min,65 ℃,15min,4 ℃.
S52, joint connection:
the reaction system was configured on ice: the end repair products, 65uL,rapid ligation buffer2, 25uL,rapid DNAligase,5uL,DNA adapter x,5uL, were gently mixed by pipetting 10 down and centrifuged for 3-5s transiently for PCR reaction under the following conditions: the heat cover was temporarily stored at 105℃at 20℃for 15min at 4 ℃.
S53, magnetic bead purification of a joint connection product:
VASHTS DNA clear Beads equilibrate to room temperature in advance for 30min, shake 5s uniformly mixing the Beads with a front vortex, absorb 100uL Beads to 100uL joint connection products (volume ratio 1:1), shake 5s uniformly mixing, incubate for 5min at room temperature, instantly centrifuge for 3-5s, place the magnetic Beads and liquid on a magnetic rack, stand for 5min, carefully absorb supernatant after the solution is clarified, rinse the Beads twice with 200uL freshly prepared 80% ethanol, incubate for 30s at room temperature, carefully absorb supernatant, uncover air dry the Beads for 5min until no ethanol remains, take the PCR tube out of the magnetic rack, perform elution: adding 25uL of eluent (10 mM Tris-HCl, pH 8.0-8.5), shaking with vortex for 5s, mixing, incubating at room temperature for 2min, centrifuging for 3-5s instantaneously, separating magnetic beads and liquid with a magnetic rack, standing for 5min, carefully sucking 23uL of supernatant into a new EP tube after the solution is clarified, and preserving at-20deg.C for a long time.
S6, cloning a vector and controlling quality of a library:
s61, adding a carrier homology arm:
the vector homology arms were added by PCR reaction. PCR reaction 25uL: KAPA HIFI Hotstart Mix,12.5uL, adapter-F primer, 0.5uL, adapter-R primer, 0.5uL, adapter ligation product, 11.5uL.
S62, recycling a PCR product PAGE gel:
all 25uL PCR products are loaded into spot sample holes, electrophoresis is carried out for 30min under 120V voltage, and target bands in the range of 110-150bp are cut for recovery and purification. The experimental procedure is detailed in S41.
S63, cloning a vector: kit Clone Express II one step cloning kit, vazyme, C112
The optimal dosage is as follows: carrier amount (0.03 pmol) = (0.02 x carrier base number) ng
Amount of insert (0.06 pmol) = (0.04 x number of fragment bases) ng
Gently beating for 10 times, mixing, and instantly centrifuging for 3-5 times. Incubation was carried out in a PCR apparatus at 37℃for 30min. Temporary storage is carried out at 4 ℃.
S64, transformation and fungus culture:
thawing competent cells on ice, adding 10uL of recombinant product into 100uL of competent cells, performing heat shock in a water bath at 42 ℃ for 45s, immediately cooling on ice for 2-3min, adding 900uL of LB (without antibiotics), resuscitating at 37 ℃ for 1h (200-250 rpm) in a constant temperature shaking table, adding 30mL of LB into each tube, 60uL of Amp, and shaking at 37 ℃ for 12-16h in a constant temperature shaking table at 200 rpm.
S65, plasmid extraction:
the bacterial liquid is centrifuged for 10min at the room temperature of 5000 Xg, bacterial precipitate is collected, supernatant is carefully removed, 250uL of Solution I/RNase A is added, suspension bacterial liquid is blown up and down by vortex or a pipetting gun, 250uL of Solution II is added, and the mixture is gently inverted and evenly mixed for 4-6 times, thus obtaining clear lysate. Incubation was performed at room temperature for 2min, 125uL of pre-chilled BufferN3 (placed on ice in advance), mixing was reversed several times until white flocculent precipitate appeared, centrifuging at 12000×g 25 ℃ for 10min, carefully transferring the supernatant to a clean 1.5mL centrifuge tube, and adding 0.1 volumes of ETR Solution. Mixing for 7-10 times, and incubating on ice for 10min, wherein the incubation process can be reversed several times. Incubation was carried out at 42℃for 5min, and the lysate was again clouded and centrifuged at 12000 Xg at 25℃for 3min. Transferring the supernatant to a new 1.5mL centrifuge tube, adding 0.5 times of 96% -100% ethanol (room temperature), mixing for 6-7 times, incubating for 1-2min at room temperature, collecting the mixed solution, adding into a clean HiBindTM DNA Mini column assembly and a 2mL collecting tube, centrifuging at 10000×g at room temperature for 1min, passing through the column, and discarding the filtrate. The column was washed with 500uL Buffer HB and centrifuged as above. The column was washed twice with 700 a uL DNAWash Buffer and centrifuged as above. The filtrate was discarded, the empty column was returned to the centrifuge tube, centrifuged at maximum speed (. Gtoreq.13000 Xg) for 2min, and the column was dried. The column was placed in a new 1.5mL centrifuge tube, 80uLEndotoxin-Free Elution Buffer was applied to the column and centrifuged at maximum speed (. Gtoreq.13000 Xg) for 1min to elute the plasmid.
S66, library preparation:
amplifying target sequences containing inserts by using the recombinant plasmid library as a template, and amplifying primer information: pMX-TY1, TGCAGGTGCCAGAACATTTC, pMX-TY2, GTGGCTTTACCAACAGTACC. And (3) recovering and purifying products with the size of 209-244bp by using a PAGE gel, wherein the gel recovery flow is shown as S62, constructing a DNA sequencing library by taking the PCR recovery product as input DNA, recovering and purifying target fragments by using agarose gel, wherein the library fragment size is 332-367bp. The library construction procedure is shown in S41.
The main technical parameters and experimental results in this embodiment are as follows:
1. ultrasonic disruption of genome and MNAse cleavage:
1ug of DNA is taken to carry out ultrasonic disruption of the product (100-200 bp), mnase digestion (4 ℃,30min,100uL system) is carried out, and the working concentration of Mnase is 1U/uL.
2.15-50bp short fragment DNA product recovery:
the digested product was first column purified using Oligo Clean & Concentrator Kit at 190.8ng yield, then subjected to 12% PAGE gel electrophoresis and fragment recovery in the 15-50bp range (FIG. 1) at 74.88ng yield.
3.15-50bp short fragment DNA ligation adapter:
the end repair of the gel recovery product was added with A, then adapter ligation (Novain N805) was performed, the concentration of the adapter stock solution was 200uM, 100uL of the ligation reaction system was ligated, and the amount of adapter was calculated according to the following formula. The average length of DNA is about 33bp. The ratio of the number of Adapter to input DNA molecules was 50:1.
the approximate molecular weight of nucleic acid is calculated by the formula:
molecular weight of dsDNA (g/mol) = (number of nucleotides x 607.4) +157.9, the molecular weight values of dsDNA in this example are shown in table 3.
TABLE 3 dsDNA molecular weight values for example 1
| Nucleotide number | Molecular weight (g/mol) | Molecular weight (ng/pmol) |
| 33 | 20,202.10 | 20.20 |
Adapter ligation product addition vector homology arm:
the adapter ligation product was subjected to addition of a vector homology arm by means of PCR. The F and R primers and the adapter have 34bp complementary sequences (marked as a whole), and the 5' end of the primers contains 15bp carrier homologous sequencesIndication). F:R：PCR reactions were performed with 10 cycles. The PCR products were subjected to 12% PAGE, and fragments ranging from 110 to 150bp were cut and purified (FIG. 2) in 93.6ng yield. The fragment size is shown in detail in FIG. 3.
5. Constructing a plasmid library and controlling quality:
the backbone vector was subjected to double cleavage with restriction enzymes XhoI and HindIII, 50uL cleavage system, and cleavage at 37℃for 1h (FIGS. 4A, 4B). The PCR purified product was ligated with a linearized vector by homologous recombination. The vector size was 5800bp, the amount was 112ng, the insert size was about 150bp, and the amount was 6ng.20ul of the ligation system: pmx-mp-mCherry linearization vector: 112ng
An insert: 6ng
5x CEⅡBuffer：4uL
ExnaseⅡ：2uL
H 2 O：to 20uL
The ligation product was transformed into 200uL E.coli competent, and the plasmid was extracted after 15h of culture (FIG. 4C). 100ng of plasmid library is taken, target fragments are amplified by using pMX-TY1 and pMX-TY2 primers, and a PAGE gel is performed to recover and purify fragments of 200-250bp (figure 5), and the size of the fragments is shown in figure 3. And constructing a DNA sequencing library by taking the PCR recovery product as input DNA, wherein the size of a library fragment is 332-367bp, recovering and purifying a target fragment by using agarose gel (FIG. 6A), constructing a library, performing S5 on the library construction flow, and performing library quality control by using 2100 (FIG. 6B). The library structure is as follows:
adapter-TGCAGGTGCCAGAACATTTCTCTGGCCTAACTGGCCGGTACCTGAGCTCGCTAGCCACAC TCTTTCCCTACACGACGGTTGCTTGATCCT-DNA fragments(15-50bp)-AGATCGGAAGAGCACACGTCTG AACTCCAGTCACTAGACACTAGAGGGTATATAATGGAAGCTCGACTTCCAGCTTGGCAATCCGGTACTGTTGGTAAAGCCAC-adapter. The DNA insert is 15-50bp, wherein the part marked as "is the adapter added when constructing the vectorPrimer binding sites.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and are not limiting. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the appended claims.
Claims (8)
1. A method of constructing a genomic short fragment library, comprising the steps of:
s1, extracting whole genome of a cell;
s2, genome fragmentation and product recovery and purification: breaking the whole genome extracted in the step S1 by using an ultrasonic instrument and recovering a broken product;
s3, mnase digestion and product recovery: carrying out Mnase digestion on the broken product in the step S2 and recovering digested products;
s4, electrophoresis and glue recovery: electrophoresis is carried out on the enzyme digestion product obtained in the step S3, and fragments with the range of 15-50bp are cut and recovered;
s5, terminal repair and joint connection: performing end repair and A addition on the DNA fragment recovered in the step S4, and performing adapter ligation on the DNA fragment subjected to end repair to obtain an adapter ligation product;
s6, vector cloning and library construction: adding a carrier homology arm into the adapter connection product obtained in the step S5, then carrying out carrier cloning to obtain a recombinant product, carrying out transformation culture on the recombinant product, extracting recombinant plasmid, carrying out amplification by taking the recombinant plasmid as a template, and constructing a DNA sequencing library by taking the amplified product as input DNA.
2. The method of constructing a genomic short fragment library according to claim 1, wherein step S1 comprises the steps of:
s11, digestion and lysis of cultured cells;
s12, purifying by column: purifying the digested and cracked digestive juice by using an adsorption column to obtain purified genome DNA.
3. The method of constructing a genomic short fragment library according to claim 1, wherein step S2 comprises the steps of:
s21, breaking the whole genome DNA of the cells by using an ultrasonic instrument;
s22, purifying the broken DNA fragments by using an adsorption column to obtain purified broken products.
4. The method of claim 1, wherein step S4 is performed by TBE PAGE.
5. The method of constructing a genomic short fragment library according to claim 1, wherein step S5 comprises the steps of:
s51, repairing the tail end: performing END repair on the DNA fragment recovered in the step S4 by using an END prepmix, and adding A;
s52, joint connection: performing adapter ligation on the DNA fragments with the repaired tail ends by using a DNA adapter to obtain an adapter ligation product;
s53, magnetic bead purification of a joint connection product: and (3) performing magnetic bead purification on the adapter ligation product to obtain a purified adapter ligation product.
6. The method of constructing a genomic short fragment library according to claim 1, wherein step S6 comprises the steps of:
s61, adding a carrier homology arm: adding a carrier homology arm to the adapter ligation product by a PCR reaction;
s62, recovering PCR products: cutting a target band in a range of 110-150bp, and recovering and purifying;
s63, cloning a vector: connecting and amplifying the adapter connecting product with a carrier to obtain a recombinant product;
s64, transformation culture: allowing the recombinant product to enter competent cells to complete transformation and culturing the transformed cells;
s65, plasmid extraction: extracting recombinant plasmid from the cultured transformed cells;
s66, library construction: amplifying a target sequence containing an insert fragment by taking the recombinant plasmid as a template, recovering and purifying a product with the size of 209-244bp, and constructing a DNA sequencing library by taking the expanded and recovered product as input DNA, wherein the size of a library fragment is 332-367bp.
7. The method of constructing a genomic short fragment library according to any one of claims 1 to 6, wherein the genome is of an animal, plant or microorganism.
8. The method of claim 7, wherein the genome is of an animal.
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