CN105442051A - Screening method of gene library - Google Patents
Screening method of gene library Download PDFInfo
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- CN105442051A CN105442051A CN201410505278.4A CN201410505278A CN105442051A CN 105442051 A CN105442051 A CN 105442051A CN 201410505278 A CN201410505278 A CN 201410505278A CN 105442051 A CN105442051 A CN 105442051A
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Abstract
The invention discloses a screening method of a gene library. The method includes the steps of: extracting the plasmids of a plurality of library pools or library monoclones in the gene library; interrupting the plasmids, and constructing fragment library for sequencing; subjecting the fragment library to sequencing to obtain a sequencing fragment read sequence; and comparing a known objective sequence with the sequencing fragment read sequence to obtain the positive library pool or library monoclone corresponding to the fragment read sequence in comparison. With good accuracy, the method provided by the invention has greatly shortened operation time, and for the library pools or library monoclines retrieved according to needs, re-experiment is unnecessary, and only data comparison is needed again. And the method is simple and effective.
Description
Technical field
The present invention relates to technical field of molecular biology, particularly relate to a kind of screening method of gene library.
Background technology
Gene library refers in a kind of carrier colony, and the set of each cloned sequence of a certain biological DNA of random collecting, contains the full detail of these species in theory.Gene library comprises genomic library and cDNA library.Gene library can make biological genetic information store with stable recombinant form, the more important thing is that it is the main path of separating clone goal gene, studies all significant to specific gene in genome or full-length genome.
The carrier that tradition construction of gene library adopts generally mainly contains plasmid (Plasmid), coemid (Cosmid), lambda particles phage, BAC carrier (bacterial artificial chromosome carrier) and yac vector (yeast artificial chromosome's carrier) etc.Wherein plasmid, coemid, lambda particles phage are due to bearer capabilities little (<45K), the library built up is general according to demand preserves with the form of pond, library (pool), and BAC and yac vector capacity are at more than 100K, the library built up is then general preserves with monoclonal form.And this vector stabilisation for alternative Cosmid of Fosmid is good, can singly to copy existence in Host Strains, reach high copy through certain induction, and have randomness good, build the advantages such as storehouse is simple, become novel vector popular at present.
After gene library is built up, for obtaining target clone, need screen library, and library screening mainly contains two kinds of methods at present: PCR sieve method and molecular hybridization.The appearance of round pcr adds a new tool to the screening of clone.If the length of known aim sequence and the sequence at two ends, then can design and synthesis pair of primers, with the DNA of transformant gained for template increases, expect the PCR primer of length if can obtain, then this transformant just may contain aim sequence.And the method that molecular hybridization is commonly used is duplicated on nitrocellulose membrane by the bacterium colony grown after conversion, use alkali cracking bacterium, the DNA of bacterium colony release is just adsorbed on film, then hybridizes with the nucleic acid probe incubation of mark, and nucleic acid probe is just combined on the bacterium colony DNA containing aim sequence not by wash-out.Nucleic acid probe can use radioisotope labeling, the bacterium colony group using radiation autography method combining radioactive nuleus acid probe indicates, nucleic acid probe also can mark by non-radioactive substance, normally present indicating positions through color, so just the bacterium colony containing aim sequence can be picked out.
Above two kinds of methods all need to enter specific experiment operation with the primer synthesized or probe to library, experimental procedure is loaded down with trivial details, and because reagent or the factor such as artificial cause false positive or false negative, and when having changed different primers or probe, need experiment be re-started.
Summary of the invention
The invention provides a kind of screening method of gene library, the method accuracy is good, the operating time shortens greatly; And for the pond, library needing to transfer or library mono-clonal, without the need to again testing, only need re-start comparing, the method is simply effective.
A screening method for gene library, comprises the steps:
Extract the monoclonal plasmid in pond, multiple library or library of gene library;
Interrupt plasmid, build the frag-ment libraries of order-checking;
Frag-ment libraries is checked order, obtains the order-checking section of reading sequence; With
By known aim sequence and the order-checking section of reading sequence alignment, obtain pond, positive library that the section of the reading sequence pair in comparison answers or library mono-clonal.
Method of the present invention realizes gene library screening based on order-checking and comparison, is different from traditional PCR sieve method and molecular hybridization completely.Method of the present invention can be screened the gene library of preserving with pond, library or library monoclonal in form.Although shown in one embodiment of the present of invention is screen the gene library of preserving with pond, library form, based on principle of the present invention and total design, certainly can screen the gene library of preserving with library monoclonal in form.
As preferred version of the present invention, interrupt plasmid, the step building the frag-ment libraries of order-checking specifically comprises: interrupt plasmid with interrupting instrument; End reparation is carried out to the fragment interrupted; Nick translation reaction is carried out with jointing.
As preferred version of the present invention, after nick translation reaction, the fragment of predetermined length is selected to be used for order-checking.The effect of this step is, by selecting fragment length, improves the validity of order-checking.Because, although also can pass through the generation arranging the fragment realizing a certain extent length interrupting instrument working parameter, but the fragment after interrupting still distributes in a wider scope, and too short or long fragment is all unfavorable for producing effective sequencing data, therefore preferably carries out this step.
As preferred version of the present invention, predetermined length is 100 ~ 5000bp; Preferably 100 ~ 2000bp; More preferably 100 ~ 500bp; Most preferably 200 ~ 250bp.
As preferred version of the present invention, select predetermined length fragment by agarose gel electrophoresis and cut glue reclaim carry out.After agarose gel electrophoresis, nucleic acid fragment is the distribution of disperse shape, with nucleic acid standard molecular weight for reference to cutting the gel of predetermined length scope, can obtain the fragment of predetermined length after column purification.
As preferred version of the present invention, the order-checking degree of depth of order-checking is more than 1.5; Preferably more than 2.0; Most preferably 2.0.So-called " the order-checking degree of depth ", refers to the ratio of base total amount and the Genome Size checking order and obtain, and it is one of index evaluating order-checking amount.Be a positively related relation between the order-checking degree of depth and genome coverage, the error rate that order-checking brings or false positive results can decline along with the lifting of the order-checking degree of depth.In the method for the invention, the order-checking degree of depth about 2.0 can meet the requirement of gene library screening, compares other method economical and efficient of the present invention.
As preferred version of the present invention, gene library is Fosmid library, Cosmid library, BAC library or YAC library.Wherein, Fosmid library and Cosmid library are typically with the gene library that pond, library form is preserved; And BAC library or YAC library are typically with the gene library that library monoclonal in form is preserved.Certainly, of the present invention being not limited to is screened above-mentioned several gene library, but is applicable to screen any gene library of preserving with pond, library or library monoclonal in form.
As preferred version of the present invention, gene library is Fosmid library, and the storage capacity in Fosmid library is 10 times of genomic dna.All goal gene can be contained substantially in the Fosmid library that storage capacity is equivalent to 10 times of genomic dna, and storage capacity is too large may cause the waste in library and the increase of order-checking amount, the too little omission that may cause goal gene.
As preferred version of the present invention, gene library is stored in microwell plate with the monoclonal form in pond, library or library.Preserve gene library high-efficient simple with microtitre plate format, be convenient to high-throughput operation, screening is convenient, and generally several pieces of microwell plates can realize the preservation of whole gene library.Certainly, other modes such as EP pipe etc. is also applicable to preserving gene library.
As preferred version of the present invention, microwell plate is 96 microwell plates.
The present invention realizes the screening of gene library by order-checking and the mode of comparison, compares traditional method, without the need to a large amount of primer and probe; Avoid the false positive because experiment reagent or human users etc. cause or false negative, accuracy is high, and the operating time shortens greatly; Only need the lower order-checking degree of depth (2 × left and right) to meet requirement of experiment, and the order-checking section of reading sequence is without the need to assembling; And for the pond, library needing to transfer or library mono-clonal, without the need to again testing, only need re-start comparing, therefore the method is simply effective.
Accompanying drawing explanation
Fig. 1 is that in embodiment 1 cynomolgus monkey 10 × Fosmid library construction, DNA interrupts the detected through gel electrophoresis result figure under number of times different situations.Wherein, L1 and L2 represents low scope PFGMarker respectively; 0,100,200,300 and 400 represent that cynomolgus monkey genomic dna uses rifle head to interrupt the electrophoresis result after 0,100,200,300 and 400 time respectively.
Fig. 2 is the collection of illustrative plates of the Fosmid plasmid vector pcc2FOS used in embodiment 1 cynomolgus monkey 10 × Fosmid library construction.
Fig. 3 is the schematic diagram that cynomolgus monkey 10 × Fosmid library of embodiment 1 structure is kept in 3 96 orifice plates.
Fig. 4 be in embodiment 2 by agarose gel electrophoresis and cut glue reclaim carry out Piece Selection time cut glue before (left figure) and afterwards (right figure) agarose gel electrophoresis image ratio comparatively, wherein M represents DNAMarker, and 1 and 2 represent the electrophoresis result of two frag-ment libraries respectively.
Embodiment
By reference to the accompanying drawings the present invention is described in further detail below by embodiment.
Embodiment 1 constructs cynomolgus monkey 10 × Fosmid library, and embodiment 2 is screened the pond, library on piece 96 orifice plates of in this library.
Embodiment 1:Fosmid library construction
The present embodiment adopts CopyControl
tMfosmidLibraryProductionKit kit reagent and explanation, construct cynomolgus monkey 10 × Fosmid library, prepared about 1.5 × 10
5individual clone ,-80 DEG C are stored in 3 96 orifice plates.Concrete library constructing method is implemented as follows:
1, interrupt: first DNA sample integrity and the condition that interrupts are groped, get 3 μ gDNA, do respectively and interrupt process (100 μ L rifle head) for 0,100,200,300 and 400 time, pulsed field gel electrophoresis detects, impulsive condition: 1% agarose gel, 0.1-40s, 6v, 16h.Adopt low scope PFGmarker as DNAMarker.If DNA is complete, the DNA master tape interrupted for 0 time should at more than 48.5K; Interrupting rational number of times should be make DNA master tape between 48.5K and 97K, interrupts result as shown in Figure 1, interrupt 200 times most suitable.
2, magnetic beads for purifying: the DNA AmpureBeads after interrupting carries out purifying, is stored in 4 DEG C of refrigerators, must be positioned over equilibrium at room temperature half hour before using after AmpureBeads packing, add the AmpureBeads of 1.8 times of sample volumes, put upside down mixing, room temperature places 10min, frequently mix gently, being placed on magnetic frame makes magnetic bead be adsorbed in tube wall, remove supernatant, add 500 μ L70% ethanol and wash magnetic bead, remove supernatant, add 500 μ L70% ethanol again and wash magnetic bead, remove supernatant, 37 DEG C of dryings are until magnetic bead starts to occur dry and cracked, add the resuspended magnetic bead of 30 μ L elution buffer, room temperature places 10min, be placed on magnetic frame, supernatant is gone to new pipe, use the resuspended magnetic bead of 30 μ L elution buffer again, room temperature places 10min, be placed on magnetic frame, supernatant is gone to new pipe.
3, end-filling: end-filling reagent and system as follows:
Above-mentioned system is hatched 2h at 25 DEG C.
4, ligation: purify with AmpureBeads, then carries out ligation, and wherein the collection of illustrative plates of Fosmid carrier pcc2FOS as shown in Figure 2.Agents useful for same and system as follows:
Gently after mixing, hatch 3h for 25 DEG C, 12 DEG C of ∝.Then, 70 DEG C of deactivation 10min.
5, Fosmid clone is packed:
A) get a tube packaging extract (MaxPlaxLambdaPackagingExtracts), after thawed on ice, draw in 25 μ L to 1.5mL centrifuge tubes, then put back to-80 DEG C of refrigerators;
B) 10 μ L are connected product to join in remaining 25 μ L packaging extracts, beat gently with wealthy rifle head, hatch 90min for 30 DEG C;
C) from refrigerator, take out 25 previous μ L packaging extracts, thawed on ice, then adds, and beats gently with wealthy rifle head, hatches 90min for 30 DEG C;
D) then adding phage dilution buffer to final volume is 1mL, adds 25 μ L chloroforms, puts upside down several times, is put in 4 DEG C (unavailable whizzers), can places 2 weeks with have gentle hands after getting rid of.
6, competent cell preparation and conversion:
A) preparation of competent cell: the day before yesterday of packaging, proceeds to 50mlLB substratum (containing 10mMMgSO by monoclonal cell
4), 30 DEG C, 180rpm rotating speed, incubated overnight (noting using 250mL triangular flask); Packaging was got 5mL overnight culture and was inoculated into 50mLLB substratum (containing 10mMMgSO the same day
4), 30 DEG C, 250rpm rotating speed, cultivates 120min (note use 250mL triangular flask), be placed in 4 DEG C for subsequent use, 72h can be placed under 4 DEG C of conditions at most;
B) transform: get 10 μ L and pack products and add in the competent cell that 90 μ L have prepared, hatch 20 minutes for 37 DEG C; Get on solid LB media that 100 μ L are coated on containing final concentration 12.5 μ g/mL paraxin after having hatched, 30 DEG C of overnight incubation.
7, pond, Fosmid library is preserved: count the clone on all flat boards, the flat board that sum about 3000 is cloned is shared 2mL LB liquid medium (containing 12.5 μ g/mL paraxin) rinse, collect elutriant, this elutriant and pond, library are added sterile glycerol to final concentration 20%, proceed to 2mL cryopreservation tube, after all completing, put into three 96 orifice plates (as shown in Figure 3) successively, carry out mark ,-80 DEG C are stored in refrigerator.
Embodiment 2:Fosmid library screening
1, upgrading grain: take out frozen pond, Fosmid library (first piece of 96 orifice plate) from-80 DEG C of refrigerators, room temperature fully puts upside down mixing after melting, 50 μ L bacterium liquid are got from every hole, add 6mL containing 1% paraxin LB substratum, 37 DEG C are spent the night and shake bacterium, and secondary daily Invitrogen large fragment test kit extracts pond, library plasmid, add 37 DEG C of half an hour of RNA enzyme, and purifying removes enzyme wherein and small molecules etc. as far as possible, be dissolved in not containing in the water of nuclease.
2, pond, library plasmid builds storehouse:
A) interrupt: get pond, library plasmid 1 μ g, be diluted to 80 μ L, use CavorisLE220 to interrupt instrument to interrupt, interrupt parameter: space factor (dutyFactor) 21, specific resistance (PIP, W) 500, outburst frequency (cyclesperBurst) 500; Temperature (BathTemperature) 7.9; Time: 20s, 30 times.
B) end reparation and purifying, agents useful for same and system as follows:
At 20 DEG C of reactions 30min, then magnetic beads for purifying.
C) joint connects and nick translation and purifying, agents useful for same and system as follows:
Note: sequence measuring joints P-joint and A-joint are the joint sequence that IlluminaHiSeq order-checking platform uses, and each platform has specific sequence measuring joints.
After above-mentioned system piping and druming mixing, be put in PCR instrument and run following response procedures: 25 DEG C, 15min; 72 DEG C, 5min; 12 DEG C of ∝.After having reacted, magnetic beads for purifying.
D) Piece Selection and amplification: the agarose gel electrophoresis with 2.25% and QIAquick extract test kit and cut glue recovery, result as shown in Figure 4, show and the situation of Piece Selection is carried out to wherein pond, two libraries, wherein pond, library 1 and 2 is selected respectively to the fragment of about 200 ~ 250bp size, all Piece Selection has been carried out for the pond, 96 libraries in first piece of 96 orifice plate in experiment, except 1 and 2, other does not illustrate.Cross column purification after cutting glue, purification step is with reference to QIAGEN specification sheets purification step.
E) Library Quality detects: draw the order-checking library that 4 μ L build, carry out Agilent2100 detection, object peak is about 230-330bp, and be namely available on the machine order-checking.
3, checked order in the above-mentioned order-checking library built, obtain lower machine literature data and namely to check order the section of reading sequence (reads).
4, by the aim sequence for library designs respectively with lower machine literature data reads comparison, if with in certain order-checking library comparison, can just illustrate that the pond, library of its correspondence is the positive library of this aim sequence.
The present embodiment is with rhesus monkey MHC district scaffold for reference, and choosing 79 site recombination sequences (SEQIDNO:1 ~ 79) and lower machine literature data reads comparison, to obtain corresponding pond, positive library result as shown in table 1.In follow-up homologous recombination experiments, all can recall positive monoclonal from pond, positive library.
Table 1
Above content is in conjunction with concrete embodiment further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made.
Claims (10)
1. a screening method for gene library, comprises the steps:
Extract the monoclonal plasmid in pond, multiple library or library of gene library;
Interrupt described plasmid, build the frag-ment libraries of order-checking;
Described frag-ment libraries is checked order, obtains the order-checking section of reading sequence; With
By known aim sequence and the described order-checking section of reading sequence alignment, obtain pond, positive library that the section of the reading sequence pair in comparison answers or library mono-clonal.
2. method according to claim 1, is characterized in that, described in interrupt described plasmid, the step of frag-ment libraries building order-checking specifically comprises: interrupt described plasmid with interrupting instrument; End reparation is carried out to the fragment interrupted; Nick translation reaction is carried out with jointing.
3. method according to claim 2, is characterized in that, after described nick translation reaction, selects the fragment of predetermined length to be used for order-checking.
4. method according to claim 3, is characterized in that, described predetermined length is 100 ~ 5000bp; Preferably 100 ~ 2000bp; More preferably 100 ~ 500bp; Most preferably 200 ~ 250bp.
5. method according to claim 3, is characterized in that, the fragment of described selection predetermined length is by agarose gel electrophoresis and cut glue and reclaim and carry out.
6. method according to claim 1, is characterized in that, the order-checking degree of depth of described order-checking is more than 1.5; Preferably more than 2.0; Most preferably 2.0.
7. method according to claim 1, is characterized in that, described gene library is Fosmid library, Cosmid library, BAC library or YAC library.
8. method according to claim 7, is characterized in that, described gene library is Fosmid library, and the storage capacity in described Fosmid library is 10 times of genomic dna.
9. method according to claim 1, is characterized in that, described gene library is stored in microwell plate with the monoclonal form in pond, library or library.
10. method according to claim 9, is characterized in that, described microwell plate is 96 microwell plates.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207471A (en) * | 2017-06-30 | 2019-01-15 | 深圳华大基因股份有限公司 | A kind of method and its application constructing fragment section nucleic acid library |
| CN110241189A (en) * | 2019-06-22 | 2019-09-17 | 华中农业大学 | A kind of long paired end sequencing method in DNA long fragment library |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409043A (en) * | 2010-09-21 | 2012-04-11 | 深圳华大基因科技有限公司 | Method for constructing high-flux and low-cost Fosmid library, label and label joint used in method |
| CN102409048A (en) * | 2010-09-21 | 2012-04-11 | 深圳华大基因科技有限公司 | DNA index library building method based on high throughput sequencing |
| CN102560688A (en) * | 2010-12-15 | 2012-07-11 | 深圳华大基因科技有限公司 | Novel library construction method based on illumina sequencing platform |
-
2014
- 2014-09-26 CN CN201410505278.4A patent/CN105442051A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409043A (en) * | 2010-09-21 | 2012-04-11 | 深圳华大基因科技有限公司 | Method for constructing high-flux and low-cost Fosmid library, label and label joint used in method |
| CN102409048A (en) * | 2010-09-21 | 2012-04-11 | 深圳华大基因科技有限公司 | DNA index library building method based on high throughput sequencing |
| CN102560688A (en) * | 2010-12-15 | 2012-07-11 | 深圳华大基因科技有限公司 | Novel library construction method based on illumina sequencing platform |
Non-Patent Citations (2)
| Title |
|---|
| 秦培钢: "水稻纹枯病菌Rhizoctonia solani AG1IA Fosmid 基因组文库的构件及线粒体全基因组组装分析", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 陶杰等: "《生物制药工艺技术》", 28 February 2013, 中国医药科技出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207471A (en) * | 2017-06-30 | 2019-01-15 | 深圳华大基因股份有限公司 | A kind of method and its application constructing fragment section nucleic acid library |
| CN110241189A (en) * | 2019-06-22 | 2019-09-17 | 华中农业大学 | A kind of long paired end sequencing method in DNA long fragment library |
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