CN105603535A - Kit and method for constructing DNA library - Google Patents
Kit and method for constructing DNA library Download PDFInfo
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- CN105603535A CN105603535A CN201610056639.0A CN201610056639A CN105603535A CN 105603535 A CN105603535 A CN 105603535A CN 201610056639 A CN201610056639 A CN 201610056639A CN 105603535 A CN105603535 A CN 105603535A
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- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
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Abstract
The invention provides a kit and method for constructing a DNA library. The kit comprises an enzymic reagent, a buffer solution and a joint combination, wherein the enzymic reagent comprises a first enzyme mixed liquid, T4DNA ligase and a second enzyme mixed liquid, and the first enzyme mixed liquid is formed by mixing T4 Polynucleotide Kinase, T4DNA polymerase, a Klenow fragment and rTaq enzyme; the second enzyme mixed liquid is a 2X KAPA HiFi HotStart pre-mixed solution; the buffer solution comprises a first buffer solution and a second buffer solution, and the first buffer solution is formed by mixing a 10X T4DNA ligase buffer solution, dNTP mixed liquor and ATP; the second enzyme mixed liquid is formed by mixing ATP, Tris-HCL, MgCl2, PEG8000, Tween 20 and dithiothreitol; the joint combination comprises a joint sequence group and a tag sequence group. According to the kit, the library construction cost can be reduced and the amplification efficiency is improved.
Description
Technical field
The present invention relates to high-throughput sequencing library and build field, in particular to the kit in a kind of constructed dna libraryAnd method.
Background technology
Now general small pieces segment DNA library constructing method be mainly adopt NEB build storehouse kit, asUltraTMOr DNALibraryPrepKitforIllumina etc., these kits are applicable to all little substantiallyThe structure in fragment (180bp～500bp) library, comprises the sample of different plant species, different quality or different clip types. AndConstructed library is widely used in de novo sequencing (denovosequencing) or the order of resurveying (resequencing) etc.
The main flow process that mentioned reagent box builds library is as follows: S1: genome is carried out to fragmentation; S2: fragmentation DNA'sEnd is not flat end, therefore will fill to it (20 DEG C of 30min), adds A (65 DEG C of 30min) after filling; S3: adoptIt is U font joint (20 DEG C of 15min) that the mode that TA connects adds the fixing sequence of the preceding paragraph, adds USER enzyme that U font joint is beatenOpen (37 DEG C of 15min); S4: carry out Piece Selection with AMPureXP magnetic bead, obtain the required object fragment that adds joint; S5:Object fragment is carried out PCR enrichment, and PCR period can regulate according to template amount, general 6-15 circulation; S6: finally useAfter AMPureXP magnetic beads for purifying PCR product, get final product outbound.
But existing NEB builds storehouse kit in the shortcoming building aspect several under existing when library: (1) build Kucheng thisHigher: finished product kit price is generally somewhat expensive, the single sample of single build Kucheng this more than 200 yuan; (2) amplification efficiency ratioLower: the especially lower sample of sample size, cannot carry out successfully outbound; (3) purification step is easily made mistakes: flow in the storehouse of building of NEBJourney in carrying out Piece Selection conventionally through 2 step magnetic beads for purifying (going respectively large fragment and the mode of removing small fragment), the 1st stepPurifying is to stay supernatant solution to remove the large fragment being adsorbed on magnetic bead, and the 2nd step is that supernatant removes small fragment, 2 step magnetic beads for purifyingDifferent operating easily obscure; (4) magnetic beads for purifying is selectively poor: the fragment that magnetic bead is selected is wider, meeting in wide fragmentHave larger fragment, larger fragment is in a disadvantageous position with in the process of clustering at follow-up PCR, finally may cause numberAccording to output deficiency; (5) experiment flow is relatively loaded down with trivial details: the joint in NEB kit is u shaped connector, is adding after joint and can increaseThe process that u shaped connector is become breeches joint by one step interpolation USER enzyme just can be carried out follow-up PCR enrichment, and experiment flow is relatively numerousTrivial.
Therefore, even if existing kit is having many advantages aspect structure library, but still there is above-mentioned many defects, because ofAnd be still difficult to meet on market to the diversified demand of DNA library construction.
Summary of the invention
Main purpose of the present invention is to provide kit and the method in a kind of constructed dna library, to solve prior artIn the lower problem of kit amplification efficiency.
To achieve these goals, according to an aspect of the present invention, provide the kit in a kind of constructed dna library,This kit comprises: enzyme reagent, and enzyme reagent comprises that first enzyme mixes the mixed liquid of liquid, T4DNA ligase and second enzyme, first enzyme is mixedLiquid is mixed by T4 Polynucleotide kinases, T4DNA polymerase, Klenow fragment and rTaq enzyme; The mixed liquid of second enzyme is 2XKAPAHiFiHotStart premixed liquid; Buffer solution, buffer solution comprises mixed corresponding the first buffer solution of liquid of first enzyme and T4Corresponding the second buffer solution of DNA ligase, the first buffer solution by 10XT4DNA ligase buffer solution, dNTP mixed liquor andATP mixes; The second buffer solution is by ATP, Tris-HCl, MgCl2, PEG8000, Tween20 and dithiothreitol (DTT) mixForm; And splice combinations, splice combinations comprises joint sequence group and sequence label group.
Further, in the mixed liquid of first enzyme, the kinase whose working concentration of T4 Polynucleotide is 0.05～0.2U/ μ L, T4DNAThe working concentration of polymerase is 0.02～0.04U/ μ L, and the working concentration of Klenow fragment is 0.01～0.03U/ μ L, rTaq enzymeWorking concentration be 0.02～0.04U/ μ L.
Further, in the first buffer solution, the working concentration of 10XT4DNA ligase buffer solution is 0.8X～2X, dNTPWorking concentration 0.3～the 0.6mM of mixed liquor; The working concentration of ATP is 1.5～2.2mM.
Further, in the second buffer solution, the working concentration of ATP is 1.5～3mM, and the working concentration of Tris-HCl is0.04～0.08M，MgCl2Working concentration be 0.01～0.03M, the working concentration of PEG8000 is 5%～10wt%, Tween20 working concentration is 1v%～4v%, and the working concentration of dithiothreitol (DTT) is 0.001～0.003M.
Further, the working concentration of T4DNA ligase is 200～800U/ μ L; 2XKAPAHiFiHotStart is pre-The working concentration of mixed liquid is 1X.
Further, splice combinations forms breeches joint; Preferably joint sequence group is by the first joint sequence and the second joint orderRow mix according to equimolar ratio, and sequence label group is mixed according to equimolar ratio by sequence label and universal primer.
Further, the first joint sequence is shown in SEQIDNO:1GATCGGAAGAGCNNNNNNNNGAACTCCAGTCAC, the second joint sequence is: shown in SEQIDNO:2ACACTCTTTCCCNNNNNNNNGCTCTTCCGATCT, the N in SEQIDNO:1 and SEQIDNO:2 is in A, T, C or GAny; Sequence label comprises the second label shown in the first sequence label shown in SEQIDNO:3, SEQIDNO:4The 4th sequence label shown in the 3rd sequence label shown in order, SEQIDNO:5, SEQIDNO:6, SEQIDNO:7 instituteThe 6th sequence label shown in the 5th sequence label, the SEQIDNO:8 showing, the 7th sequence label shown in SEQIDNO:9,The 9th sequence label and SEQIDNO:12 shown in the 8th sequence label shown in SEQIDNO:10, SEQIDNO:11The tenth shown sequence label; Universal primer is the sequence shown in SEQIDNO:13.
To achieve these goals, according to an aspect of the present invention, provide a kind of above-mentioned any kit to buildThe method in DNA library, the method comprises: the genomic DNA of sample is carried out to fragmentation, obtain DNA fragmentation; Utilize first enzyme mixedLiquid and the first buffer solution carry out end reparation and add A DNA fragmentation, obtain repairing fragment; Utilize T4DNA ligase, second to delayRush liquid and joint primer sets and carry out joint connection to repairing fragment, obtain belt lacing fragment; Utilize the mixed liquid of second enzyme and labelSequence set is carried out PCR enrichment to belt lacing fragment, obtains DNA library.
Further, obtaining after the step of belt lacing fragment, and before belt lacing fragment is carried out to PCR enrichment,Method also comprises the step of belt lacing fragment being carried out to purifying.
Further, the step of purifying comprises the step of carrying out successively magnetic beads for purifying and Purified in electrophoresis.
Apply technical scheme of the present invention, by selecting commercially available T4 Polynucleotide kinases, T4DNA polymerase, KlenowFragment is also mixed into the mixed liquid of first enzyme, then mixed with commercially available 10XT4DNA ligase buffer solution, dNTP mixed liquor and ATPClose the first buffer solution forming and be used in conjunction with, not only can realize equally the end reparation of fragmentation DNA and add A, and reduceConstruction cost. And adopt commercially available T4DNA ligase and the second buffer solution of being formed by mentioned reagent to the DNA after repairing withAnd joint carries out joint connection, can improve joint joint efficiency. Finally, utilize 2XKAPAHiFiHotStart premixed liquidTo connecting the enrichment of increasing of the product of joint, amplification efficiency is high. Thereby, adopt mentioned reagent box of the present invention, not only canReduce library construction cost, and can improve amplification efficiency.
Brief description of the drawings
The Figure of description that forms the application's a part is used to provide a further understanding of the present invention, of the present invention showingMeaning property embodiment and explanation thereof are used for explaining the present invention, do not form inappropriate limitation of the present invention. In the accompanying drawings:
Fig. 1 shows middle according to a preferred embodiment of the invention DNA library construction flow chart;
Fig. 2 shows the testing result figure in DNA library constructed in a kind of preferred embodiment of the present invention; And
Fig. 3 shows the testing result figure in another DNA library constructed in a kind of preferred embodiment of the present invention.
Detailed description of the invention
It should be noted that, in the situation that not conflicting, the feature in embodiment and embodiment in the application can phaseCombination mutually. Describe below with reference to the accompanying drawings and in conjunction with the embodiments the present invention in detail.
It should be noted that, various commercial reagent of the present invention do not comprise on existing market whole for library constructionCertain single agents in cover kit or kit, and refer to the reagent of selling with independent form.
In the present invention, M is writing a Chinese character in simplified form of molar concentration mol/L, and mM is writing a Chinese character in simplified form of molar concentration mmol/L, and μ M is molar concentrationμ mol/L writes a Chinese character in simplified form.
10XT4DNA ligase buffer solution is 700mmTris-HCl (PH8.0), 100mmMgCl2 and 50mmDTT;Wherein, the meaning of working concentration 1X is: 70mMTris-HCl (PH8.0), 10mMMgCl2 and 5mMDTT; The work of 0.5X is denseThe meaning of degree is: 35mMTris-HCl (PH8.0), 5mMMgCl2 and 2.5mMDTT. Due in the time carrying out reagent configuration, useBe business-like 10XT4DNA ligase buffer solution, therefore, that in the reagent of using below, mention is exactly 10XT4DNA ligase buffer solution.
The working concentration of 2XKAPAHiFiHotStart premixed liquid refers to KAPAHiFiStandard by the meaning of 1XorHotStartDNAPolymerase(0.5U/μL)，1XKAPAHiFiFidelityBuffer，1XKAPAHiFiGCBuffer, 0.5mMMgCl2,0.5mMdNTPMix is (due to KAPAHiFiFidelityBuffer and KAPAHiFiGCBuffer is KAPA company oneself preparation, therefore concrete composition the unknown); 2XKAPAHiFiHotStart premixed liquidThe meaning KAPAHiFiStandardorHotStartDNAPolymerase (0.25U/ μ L) of 0.5X for working concentration,0.5XKAPAHiFiFidelityBuffer，0.5XKAPAHiFiGCBuffer，0.25mMMgCl2，0.25mMdNTPMix。
As background technology part is mentioned, there is the high and amplification effect of cost in existing kit in the time of constructed dna libraryRate is low, but existing kit is as the protection product of each businessman, the composition of the reagent composition in its kit of selling andRatio is all maintained secrecy, thereby although the kit composition of different manufacturers may be more similar, the effect after using respectively hasDifferent. Inventor is expensive for commercially available kit finished product, and utilizes the defects such as effect is unstable, attempts to research and develop voluntarily a kind of being suitable forIn the kit of DNA library construction, first, inventor enters respectively various reagent used in the each reactions steps of library constructionGone purchase, and distributed rationally, then taking through the sample DNA of over-richness and quality testing after experiment sample has carried outContinuous library construction process.
The process of specific experiment is as follows: the genomic DNA after detecting is carried out to fragmentation, and the DNA of fragmentation carries out electrophoresisSmear detects, if, at 230bp, can meet follow-up database work within the scope of 300bp and 500bp; Utilize the examination of autogamyAgent is carried out end reparation and adds A the DNA of fragmentation; Add and utilize after A the reagent of autogamy and own synthetic joint to adding AFragment carry out the connection of joint; Ago-Gel with 1%～1.5% carries out electrophoresis, and after electrophoresis, cut glue and carry out Piece Selection,Reclaim kit with the Ago-Gel of sky root and carry out glue recovery; After the object fragment reclaiming with own synthetic primer and KAPAHiFiHotStartReadyMix (2X) carries out pcr amplification, conventionally the period of amplification according to initial amount number determine, oneAs 6-10 circulation; PCR product gets final product outbound after XP magnetic bead carries out purifying.
Finally, inventor detects constructed library, and testing result shows: on amplification efficiency, be better than finished product examinationAgent box is the 150-200% of the amplification efficiency of NEB kit; The GC content in constructed library more approaches the real GC of speciesContent. And avoided selecting the mode of fragment with magnetic bead, employing be to cut the mode that glue reclaims to carry out Piece Selection, selectionSheet segment limit is narrower, thereby has avoided in selected scope the relatively large fragment more difficult problem of clustering, and has solved in libraryCompared with the problem of large fragment data output deficiency.
On the basis of above-mentioned result of study, in order to reduce the cost of DNA library construction, a kind of typical real in the present inventionExecute in mode, the kit in a kind of constructed dna library is provided, this kit comprises: enzyme reagent, buffer solution and joint groupClose, wherein, enzyme reagent comprises that first enzyme mixes the mixed liquid of liquid, T4DNA ligase and second enzyme, and the mixed liquid of first enzyme is by T4 poly coreAcid kinase, T4DNA polymerase, Klenow fragment and rTaq enzyme composition; The mixed liquid of second enzyme is 2XKAPAHiFiHotStart premixed liquid; Buffer solution comprises mixed corresponding the first buffer solution of liquid of first enzyme and T4DNA ligase corresponding theTwo buffer solutions, the first buffer solution is made up of 10XT4DNA ligase buffer solution, dNTP mixed liquor and ATP; The second buffer solutionBy ATP, Tris-HCl, MgCl2, PEG8000, Tween20 and dithiothreitol (DTT) (DTT) composition, splice combinations comprises jointSequence set and sequence label group.
Mentioned reagent box of the present invention, by selecting commercially available T4 Polynucleotide kinases, T4DNA polymerase, Klenow sheetSection is also mixed into the mixed liquid of first enzyme, then mixes with commercially available 10XT4DNA ligase buffer solution, dNTP mixed liquor and ATPThe first buffer solution forming is used in conjunction with, and not only can realize equally the end reparation of fragmentation DNA and add A, and reducing structureBuild up this. And adopt commercially available T4DNA ligase and the second buffer solution of being formed by mentioned reagent to the DNA after repairing andJoint carries out joint connection, can improve joint joint efficiency. Finally, utilize 2XKAPAHiFiHotStart premixed liquid pairThe product that the connects joint enrichment of increasing, amplification efficiency is high. Thereby, adopting mentioned reagent box of the present invention, not only can fallLow library construction cost, and can improve amplification efficiency.
In the mixed liquid of first enzyme in mentioned reagent box, the consumption of each component enzymes can carry out proportioning according to existing ratio, alsoCan on the basis of existing proportioning, carry out rationally adjusting and optimize to obtain the mixed liquid of the better enzyme of remediation efficiency. In one of the present inventionIn preferred embodiment, in the mixed liquid of first enzyme, the kinase whose working concentration of T4 Polynucleotide is that 0.05～0.2U/ μ L, T4DNA are poly-The working concentration of synthase is 0.02～0.04U/ μ L, and the working concentration of Klenow fragment is 0.01～0.03U/ μ L, rTaq enzymeWorking concentration be 0.02～0.04U/ μ L. Have to repair according to the mixed liquid of the formulated first enzyme of such working concentration and mendFlat efficiency is high, and 3 ' end adds the advantage that A efficiency is high.
Equally, in the first buffer solution, the proportioning of each component can rationally be adjusted according to existing working concentration. At thisInvent in another kind of preferred embodiment, in above-mentioned the first buffer solution, the working concentration of 10XT4DNA ligase buffer solution is0.8～2X, the working concentration 0.3～0.6mM of dNTP mixed liquor; The working concentration of ATP is 1.5～2.2mM. Dense according to this workSpend the first formulated buffer solution and can effectively help the mixed liquid of first enzyme to bring into play best performance, obtain high efficiency reparation and mendFlat, 3 ' end adds A.
Equally, in above-mentioned the second buffer solution, the proportioning of each component also can be fitted on the basis of conventional working concentrationWhen adjustment obtains. In the another kind of preferred embodiment of this law, in above-mentioned the second buffer solution the working concentration of ATP be 1.5～3mM, the working concentration of Tris-HCl is 0.04～0.08mM, MgCl2Working concentration be 0.01～0.03mM, PEG8000'sWorking concentration is 5%～10% (mass concentration), and the working concentration of Tween20 is 1%～4% (volumetric concentration), two sulphur threosesThe working concentration of alcohol (DTT) is 0.001～0.003M. Can help according to the second formulated buffer solution of this working concentrationTwo enzyme mixations are brought into play best enzyme effect, and this usefulness is not subject to the impact of the first buffer solution above.
In mentioned reagent box, the working concentration of T4DNA ligase and 2XKAPAHiFiHotStart premixed liquid also canCarry out choose reasonable according to the working concentration of these two kinds of enzymes in the time carrying out DNA connection or pcr amplification in prior art. At thisIn bright another preferred embodiment, the working concentration of above-mentioned T4DNA ligase is 200～800U/ μ L; 2XKAPAHiFiThe working concentration of HotStart premixed liquid is 1X. T4DNA has good connection effect within the scope of this working concentration, canJoint is successfully connected with object fragment. The working concentration of 2XKAPAHiFiHotStart premixed liquid is within the scope of thisHave large amplification advantage, it is the highest that amplification efficiency reaches
In mentioned reagent box of the present invention, above-mentioned splice combinations is existing conventional splice combinations, such as, can be UType joint or breeches joint. Preferably adopt in the present invention breeches joint. And the joint sequence group in this breeches joint connects by firstHeader sequence and the second joint sequence mix according to equimolar ratio, sequence label group by sequence label and universal primer according to etc.Mixed in molar ratio forms. The breeches joint that adopts joint sequence group to form with sequence label group is connected and PCR enrichment for joint,There is joint efficiency and amplification efficiency is high, the high advantage of valid data amount of library output.
In above preferred embodiment, the concrete sequence of joint sequence group and sequence label group can be carried out according to actual needsDesigned, designed is synthetic or directly adopt existing sequence. In a kind of preferred embodiment of the present invention, above-mentioned first connectsHeader sequence is: the GATCGGAAGAGCNNNNNNNNGAACTCCAGTCAC shown in SEQIDNO:1, and the second joint sequence is:ACACTCTTTCCCNNNNNNNNGCTCTTCCGATCT shown in SEQIDNO:2, in SEQIDNO:1 and SEQIDNO:2N be any in A, T, C or G; Sequence label group comprises the first sequence label shown in SEQIDNO:3:CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT，SEQIDNO：4The second shown sequence label: CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCT TCCGATCT; The 3rd sequence label: CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAG shown in SEQIDNO:5TTCAGACGTGTGCTCTTCCGATCT, the 4th sequence label: CAAGCAGAAGACGGCATACGAG shown in SEQIDNO:6ATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT; The 5th sequence label shown in SEQIDNO:7:CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT，SEQIDNO：8The 6th shown sequence label: CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCT TCGATCT; The 7th sequence label: CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGT shown in SEQIDNO:9TCAGACGTGTGCTCTTCCGATCT, the 8th sequence label: CAAGCAGAAGACGGCATACGAG shown in SEQIDNO:10ATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT; The 9th sequence label shown in SEQIDNO:11:CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；SEQIDNO：12The tenth shown sequence label: CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCT TCCGATCT. Universal primer is the AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACAC shown in SEQIDNO:13GACGCTCTTCCGATCT. In above-mentioned preferred joint sequence group, only having partial sequence is complementary pairing, therefore can form YThe joint of type structure. Change u shaped connector into breeches joint, saved the process of follow-up USER enzymic digestion, simplify to a certain extentExperimental procedure, and above-mentioned each primer has the advantage that specific amplification is good, amplification efficiency is high.
In the another kind of typical embodiment of the present invention, also provide and utilized above-mentioned any kit constructed dna literary compositionThe method in storehouse, the method comprises: the genomic DNA of sample is carried out to fragmentation, obtain DNA fragmentation; Utilize the mixed liquid of first enzyme andThe first buffer solution carries out end reparation and adds A DNA fragmentation, obtains repairing fragment; Utilize T4DNA ligase, the second buffer solutionAnd joint primer sets to repair fragment carry out joint connection, obtain belt lacing fragment; Utilize the mixed liquid of second enzyme and sequence labelGroup is carried out PCR enrichment to belt lacing fragment, obtains DNA library. Implement above-mentioned by utilizing the various reagent in mentioned reagent boxThe step of DNA library construction, has saved the cost of building storehouse to a large extent, and has improved amplification efficiency, improves and builds KuchengPower.
Utilize mentioned reagent box constructed dna of the present invention library can reduce costs, improve amplification efficiency and Jian KuSuccess rate, in order further to improve the quality in constructed library and the effective dose of follow-up output data, preferably a kind of in the present inventionEmbodiment in, obtaining after the step of belt lacing fragment, and before belt lacing fragment is carried out to PCR enrichment the methodAlso comprise the step of belt lacing fragment being carried out to purifying. After the fragment of belt lacing is carried out to purifying, then carry out PCR enrichment, energyEnough reduce the amplification of non-specific product, improve the ratio of target fragment in library, so improve in follow-up output data effectivelyData volume.
Above-mentioned purification step is the conventional steps of this area, can adopt magnetic beads for purifying also can adopt Purified in electrophoresis. ?In a kind of preferred embodiment of the present invention, the step of above-mentioned purifying comprises the step of carrying out successively magnetic beads for purifying and Purified in electrophoresis.Remove in belt lacing fragment and be greater than on the basis of fragment of object length at magnetic beads for purifying, then carry out Purified in electrophoresis by object fragmentBe controlled at more accurately in length range, scope dwindle accuracy quantitative can increase follow-up upper machine time, avoid data to produceThe deficiency going out, has also solved the problem that in prior art, two step magnetic beads for purifying are easily obscured simultaneously. And the present invention adoptsBe to cut glue recovery to carry out Piece Selection, replaced original magnetic bead to select, impel object sheet segment limit narrower, avoided follow-upLarge fragment (being more than or equal to 700bp) difficulty is clustered into the problem with data output deficiency.
Further illustrate below in conjunction with specific embodiments beneficial effect of the present invention, the following example is according to Fig. 1Shown flow process is built storehouse, and fills a prescription according to the free reagent shown in table 1:
Table 1:
For the performance of free reagent in outstanding table 1, the different operating concentration range of Self-made reagent is tested, surveyEffect the building in storehouse below of examination has embodiment, and in test, the working concentration of each reagent used is as shown in table 2 below:
Table 2:
One, genomic DNA interrupts
By Qubit (fluorescent quantitation meter, lifetechnologies), genomic DNA is carried out quantitatively, DNA derives fromMicro-biological samples 24-1 (sac fungus), UN (Issatchenkia orientalis), the initial amount of these 2 samples is 1ug. Each sample standard deviation entersGone the working concentration gradient experiment of Self-made reagent, the working concentration of reagent is as above shown in table 2. 24-1 use respectively working concentration 1,2,3,4 and 5 these 5 concentration have been carried out the structure in library, respectively called after 24-1 (1), 24-1 (2), 24-1 (3), 24-1 (4) and24-1 (5); Same UN has built 5 libraries according to these 5 working concentrations, called after UN (1) respectively, UN (2), UN (3), UNAnd UN (5) (4). Then, by sample fragmentation, the condition that interrupts is according to table 3 to use ultrasonic broken washer (KQ-50E type ultrasonic cleaner)Carry out, this experiment expects that the object fragment length obtaining is 500bp.
After ultrasonic end, get DNA after 2 μ L fragmentations and carry out agarose gel electrophoresis and verify, find the sheet of broken DNABetween Duan great little 400-700bp, thereby meet the requirement of object clip size. Then, with AMPureXP magnetic bead after to fragmentationDNA carries out purifying, finally dissolves with the nuclease free water of 40ul.
Table 3.DNA broken condition
| Expection fragment (bp) | The depth of water (cm) | Transmitting power | Temperature | Time (s) |
| 500 | 5.5 | 50W | 8～15℃ | 120 |
| 300 | 5.5 | 50W | 8～15℃ | 360 |
| 230 | 5.5 | 50W | 8～15℃ | 480 |
Two, end reparation
Proportioning shown in showing by table 4 configures end reparation and adds the reaction system of A:
The reparation of table 4. end adds A reaction system
| Reagent | Volume 7 --> |
| The DNA of fragmentation | 37.5μL |
| The first buffer solution | 11μL |
| First enzyme mixes liquid | 1.5μL |
| Amount to | 50μL |
Softly mix, instantaneous centrifugal, react on PCR instrument by the response procedures shown in table 5.
The reparation of table 5. end adds A response procedures
| Temperature | Time |
| 20℃ | 30min |
| 65℃ | 30min |
| 4℃ | Finish |
After reaction finishes, place on ice at once, enter immediately next step joint coupled reaction.
Three, jointing
Press the configuration of proportioning shown in table 6 jointing coupled reaction system:
Table 6. joint coupled reaction system
| Reagent | Volume |
| T4 DNA ligase | 20μL |
| The second buffer solution | 1μL |
| Joint group | 1μL |
| Amount to | 72μL |
Softly fully mix, instantaneous centrifugal, be shown in and on PCR instrument, carry out joint coupled reaction by table 7:
Table 7. joint coupled reaction program
| Temperature | Time |
| 20℃ | 30min |
| 4℃ | Finish |
The first joint primer SEQIDNO:1 is: GATCGGAAGAGCNNNNNNNNGAACTCCAGTCAC,
The second joint primer SEQIDNO:2 is: ACACTCTTTCCCNNNNNNNNGCTCTTCCGATCT,
Joint group: the first joint primer and the second joint primer equimolar ratio example are mixed, the concentration of final every primerFor 15uM/ul. N in above-mentioned the first joint primer and the second joint primer represents any base in A, T, C or G.
Four, AMPureXP magnetic beads for purifying and cut glue reclaim
1) connect in product and add isopyknic magnetic bead to mix to joint, be then placed on magnetic frame, leave standstill pureChange, after purifying 5～10min, supernatant is removed; Then carry out wash-out with the nuclease free water of 22 μ L, obtain purifying for the first timeConnection product;
2) the connection product of above-mentioned purifying is for the first time carried out to electrophoresis, the voltage of electrophoresis on 1.5% Ago-GelFor 100V, the time of electrophoresis is 70min, then cuts the fragment (overall length of object sheet degree and joint) of 580bp-620bp;
3) carry out glue recovery with the Ago-Gel kit of day root, finally carry out wash-out with 26ul nuclease free water.
Five, PCR enrichment
Proportioning configuration PCR enrichment reaction system according to shown in table 8:
Table 8.PCR enrichment reaction system
| Reagent | Volume |
| The DNA fragmentation that joint connects | 24μL |
| KAPA HiFi HotStart premixed liquid (2X) | 25μL |
| Set of tags | 1μL |
| Amount to | 50μL |
Instantaneous centrifugal, all liq is collected to the PCR pipe end.
The first sequence label SEQID:3 is:
CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
The second sequence label SEQID:4 is:
CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
The 3rd sequence label SEQID:5 is:
CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
The 4th sequence label SEQID:6 is:
CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
The 5th sequence label SEQID:7 is:
CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
The 6th sequence label SEQID:8 is:
CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
The 7th sequence label SEQID:9 is:
CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
The 8th sequence label SEQID:10 is:
CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
The 9th sequence label SEQID:11 is
CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
The tenth sequence label SEQID:12 is
CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT；
Universal primer is that SEQIDNO:13 is:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT。
Sequence label group is that each sequence label mixes according to equimolar ratio with universal primer, the concentration of last each primerFor 10uM, in above-mentioned the first sequence label to the ten sequence labels, the 19th can be used for distinguishing each sample to the 24th bit base,Different samples is in the base sequence difference in this region. What wherein 24-1 library was used is the first sequence label to the five label ordersRow, what UN sample library was used is the 6th sequence label to the ten label series. Then, according to the amplification program shown in table 9 by upperStating PCR pipe puts into PCR instrument and increases.
Table 9.PCR amplified reaction program
After having reacted, PCR pipe is taken out, instantaneous centrifugal, all liq is collected to the pipe end, with isopyknic XP magnetic beadCarry out purifying, after purifying, carry out wash-out with 20ul nuclease free water and get final product outbound.
Six, library goes out Kuku inspection
Get 1 μ l and carry out Qubit (fluorescent quantitation meter) quantitatively, library is diluted to 2ng/ μ l with nuclease free water for subsequent use. SuitableAmount library to 2ng/ μ l, hands over supreme unit to carry out the upper machine of storehouse inspection according to Qubit concentration dilution.
Seven, library Insert Fragment and concentration detect
Library Insert Fragment size is detected with Aglilent2100 biological analyser, dense to library with q-PCR instrumentDegree detects; The testing result in two libraries that build with Self-made reagent working concentration 1 is shown in Fig. 2 and Fig. 3 (inspection in other librariesSurvey result figure similar, therefore omit). Wherein, Fig. 2 is 24-1 (1) library detection figure, and Fig. 3 is UN (1) library detection figure. From Fig. 2 andIn Fig. 3, can find out, adopt the size in the constructed library of the present invention between 580-620bp, without assorted peak, main peak obviously, nothingJoint and primer dimer, meet the Insert Fragment size that both-end order-checking requires.
Eight, the qualified upper machine of storehouse inspection
From the data output after lower machine, the cost in the constructed library of kit of the present invention and institute's output effectivelyThere is significantly difference in the cost in library that data volume and available reagent box are constructed and the valid data amount of institute's output, under specifically seeingTable 10.
Table 10:
From the above results of the present invention, the above-mentioned example of the present invention has been realized following technique effect: compare existingBased on the banking process of NEB kit, the kit that the present invention builds by the reagent of autogamy, not only can successfully build littleFragment library, and ensure that library is up-to-standard, and the reagent of autogamy only needs to buy substantially required reagent, thereby canBuild Kucheng and originally reduce to originally 1/5 current, under the higher condition of flux, greatly reduced the cost of building storehouse.
And 5 working concentrations from test: although working concentration 1 is compared working concentration 2-4 with 5 amplification efficiencyRelatively low, valid data amount accounting is also relatively low, but relatively existing business-like kit is carried aspect above-mentioned twoOn high basis, also by single sample build storehouse cost original 1/5, greatly reduce build Kucheng this. Wherein, work denseThe effect of degree 3 is best, is best proportioning; And the changing on little basis at cost of working concentration 2 and 4, amplification efficiency is phase alsoTo higher, valid data amount accounting is also relatively high, thereby is also the preferred scope of the application institute.
In addition, what the present invention adopted is breeches joint, and breeches joint can directly carry out subsequent reactions after connecting, and has avoided useThe step of USER enzyme reaction has reduced the triviality of experiment flow in certain degree. Further, this invention adopts and cuts gluePiece Selection is carried out in recovery, impels the sheet segment limit of selection to narrow, and avoids the more difficult problem of clustering compared with large fragment, after guaranteeThe output of continuous data. As can be seen here, the present invention is a kind of method that effectively builds small fragment library under lower cost, is protectingWhen demonstrate,proving library quality, having reduced again cost prepared by library, is a kind of extraordinary storehouse mode of building.
As can be seen from the above description, the above embodiments of the present invention have realized following technique effect: the present invention carriesFor a kind of method that builds small fragment library by Self-made reagent, this scheme only need be bought basic reagent, by oneself configurationBecome the mode of kit to carry out the structure in library, broken away from the dependence to available reagent box, and can ensure that Self-made reagent builds literary compositionStorehouse is higher than the constructed library of existing finished product kit valid data amount accounting in the quality of data. Therefore, the method and existingMethod is compared, and has following some advantage: (1) cost-saving (cost is only original 1/5), the present of improving gradually at fluxMy god, lower cost is more conducive to the competitiveness of enterprise. (2) amplification efficiency is high, and amplification efficiency is all greater than conventional useFinished product kit, as NEB and Kapa kit, amplification efficiency is the 150%-200% of NEB kit. (3) the GC content in libraryBetter for actual gene group, make sequencing result more accurate.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this areaArt personnel, the present invention can have various modifications and variations. Within the spirit and principles in the present invention all, to do any repairingProtection scope of the present invention changes, be equal to replacement, improvement etc., within all should be included in.
Claims (10)
1. the kit in constructed dna library, is characterized in that, described kit comprises:
Enzyme reagent, described enzyme reagent comprises that first enzyme mixes the mixed liquid of liquid, T4DNA ligase and second enzyme, described first enzyme mixes liquidMixed by T4 Polynucleotide kinases, T4DNA polymerase, Klenow fragment and rTaq enzyme; The mixed liquid of described second enzyme is 2XKAPAHiFiHotStart premixed liquid;
Buffer solution, described buffer solution comprises mixed corresponding the first buffer solution of liquid of described first enzyme and described T4DNA ligase instituteThe second corresponding buffer solution, described the first buffer solution is mixed by 10XT4DNA ligase buffer solution, dNTP mixed liquor and ATPForm; Described the second buffer solution is by ATP, Tris-HCl, MgCl2, PEG8000, Tween20 and dithiothreitol (DTT) mix andBecome; And
Splice combinations, described splice combinations comprises joint sequence group and sequence label group.
2. kit according to claim 1, is characterized in that, in the mixed liquid of described first enzyme, described T4 Polynucleotide swashsThe working concentration of enzyme is 0.05～0.2U/ μ L, and the working concentration of described T4DNA polymerase is 0.02～0.04U/ μ L, described inThe working concentration of Klenow fragment is 0.01～0.03U/ μ L, and the working concentration of described rTaq enzyme is 0.02～0.04U/ μ L.
3. kit according to claim 1, is characterized in that, in described the first buffer solution, described 10XT4DNA connectsThe working concentration of enzyme buffer liquid is 0.8X～2X, the working concentration 0.3～0.6mM of described dNTP mixed liquor; The work of described ATPConcentration is 1.5～2.2mM.
4. kit according to claim 1, is characterized in that, in described the second buffer solution, and the working concentration of described ATPBe 1.5～3mM, the working concentration of described Tris-HCl is 0.04～0.08M, described MgCl2Working concentration be 0.01～0.03M, the working concentration of described PEG8000 is 5%～10wt%, the working concentration of described Tween20 is 1v%～4v%,The working concentration of described dithiothreitol (DTT) is 0.001～0.003M.
5. kit according to claim 1, is characterized in that, the working concentration of described T4DNA ligase is 200～800U/ μ L; The working concentration of described 2XKAPAHiFiHotStart premixed liquid is 1X.
6. according to the kit described in any one in claim 1 to 5, described splice combinations forms breeches joint; Preferably, connectHeader sequence group is mixed according to equimolar ratio by the first joint sequence and the second joint sequence, and described sequence label group is by labelSequence and universal primer mix according to equimolar ratio.
7. kit according to claim 6, is characterized in that,
Described the first joint sequence is the GATCGGAAGAGCNNNNNNNNGAACTCCAGTCAC shown in SEQIDNO:1, described inThe second joint sequence is: the ACACTCTTTCCCNNNNNNNNGCTCTTCCGATCT shown in SEQIDNO:2, described SEQIDN in NO:1 and described SEQIDNO:2 is any in A, T, C or G;
Described sequence label comprises the second label shown in the first sequence label shown in SEQIDNO:3, SEQIDNO:4The 4th sequence label shown in the 3rd sequence label shown in order, SEQIDNO:5, SEQIDNO:6, SEQIDNO:7 instituteThe 6th sequence label shown in the 5th sequence label, the SEQIDNO:8 showing, the 7th sequence label shown in SEQIDNO:9,The 9th sequence label and SEQIDNO:12 shown in the 8th sequence label shown in SEQIDNO:10, SEQIDNO:11The tenth shown sequence label;
Described universal primer is the sequence shown in SEQIDNO:13.
8. a method of utilizing the kit constructed dna library described in any one in claim 1 to 7, is characterized in that instituteThe method of stating comprises:
The genomic DNA of sample is carried out to fragmentation, obtain DNA fragmentation;
Utilize the mixed liquid of described first enzyme and described the first buffer solution carry out end reparation and add A described DNA fragmentation, repairedFragment;
Utilize described T4DNA ligase, described the second buffer solution and described joint primer sets to connect described reparation fragmentHead connects, and obtains belt lacing fragment;
Utilize the mixed liquid of described second enzyme and described sequence label group to carry out PCR enrichment to described belt lacing fragment, obtain described DNALibrary.
9. method according to claim 8, is characterized in that, is obtaining after the step of described belt lacing fragment Yi JiBefore described belt lacing fragment is carried out to PCR enrichment, described method also comprises the step of described belt lacing fragment being carried out to purifying.
10. method according to claim 9, is characterized in that, the step of described purifying comprises carries out magnetic beads for purifying successivelyStep with Purified in electrophoresis.
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