CN109337897A - A kind of construction method of spot Ji enriched microsatellite library - Google Patents
A kind of construction method of spot Ji enriched microsatellite library Download PDFInfo
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Abstract
The invention discloses a kind of construction methods of spot Ji enriched microsatellite library, including digestion, the preparation of connector solution, connector connection, connection product PCR amplification, enrichment with magnetic bead and library construction；Cyclic DNA and chitin are added during extracting spot Ji genomic DNA, spot Ji Screening SSR Markers accuracy can be effectively improved and obtains efficiency；Chitin fluid is added in pcr amplification reaction liquid, can be improved the reaction susceptibility of PCR amplification to help to obtain complete high density spot Ji microsatellite marker.The utility model has the advantages that the present invention has many advantages, such as simple, efficient, at low cost, Screening SSR Markers accuracy and to obtain high-efficient, PCR reaction success rate high.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly, to a kind of building side of spot Ji enriched microsatellite library
Method.
Technical background
Spot Ji (Konosirus punctatus) is subordinate to Clupeiformes (Clupeiformes), clupeidae (Clupeidae), Ji
Subfamily (Dorosomatinae), spot Ji belong to (Konosirus), be coastal water warm pelagic fishes, be distributed widely in China,
Korea, South Korea and Japan are coastal, and in China, there is output at Bohai and Yellow Seas, the East Sea, the South Sea, are China, Japan and the Korea peninsula
The important fished species of inshore fishing.Spot Ji hands over the sediment resuspension and water-bottom interface substance of coastal ecosystems
Change important in inhibiting；It is the prey of a variety of high nutrition grade fish again simultaneously, plays and hold in marine food web frame
On open under pivotal player.Meanwhile because of its dressed fish height, fine and tender taste, delicious flavour, be by favor high-quality aquatic products it
One, people are to the demand of spot Ji also in steady-state growth in recent years.
In recent years, due to being influenced by the factors such as overfishing and other people class activities, China's spot Ji resource individual low age
The trend change, minimized is obvious, and the spot Ji fishery resources of coastal area of china are faced with the severe situation to fail comprehensively.Therefore, having must
The microsatellite molecular marker of continual exploitation spot Ji is wanted, its population genetic variations and genetic diversity water are parsed with higher resolution ratio
It is flat, so as to better Sustainable use and reasonable development this important marine fishery resources.
Microsatellite is one of the molecular genetic marker being most widely used in current population genetic and Study on Evolution, but due to
Microsatellite marker has species specificity, therefore is directed to the microsatellite research of any species, it is necessary to know that the micro- of the species defends first
Star DNA sequence dna, i.e. progress site screening.Microsatellite locus screening and its acquisition of primer are the first of microsatellite marker research
Step and a most key, time-consuming and complicated step.Numerous researchs delay to carry out a most important reason to be exactly early stage
Making time and the cost for screening microsatellite marker are relatively large, and the serviceable indicia obtained is less.In general, microsatellite locus sieves
Choosing strategy mainly has following four: first is that the traditional classical method of building small insert genomic library；It is defended second is that building is micro-
Star enriched library screens microsatellite marker；Third is that screening microsatellite locus from public database；Fourth is that using between nearly edge species
The versatility of primer, to obtain the micro-satellite primers of purpose species.Traditional classical method microsatellite positive clone rate is low, is saving
In main drive object be usually 2% or so, and utilize public database progress microsatellite locus screening, be often only limitted to DNA data compared with
More some model organisms.In addition, directly utilizing the micro-satellite primers of nearly edge species, the polymorphism in site is often bad, even
Sometimes what is amplified is not microsatellite DNA sequence, to influence the accuracy of result.Microsatellite marker has height
Polymorphism and information content abundant, codominant inheritance, stability is high, reacts the advantages that required template quantity is few and reproducible,
It is very suitable for population genetic study.
The prior art such as Authorization Notice No. is the Chinese invention patent of 102174714 B of CN, discloses a kind of full pawl of citrus
The construction method of mite enriched microsatellite library is integrated reported microsatellite locus screening strategy, is passed through using magnesphere
The series of steps such as extracting genome DNA, digestion, connector connection, enrichment with magnetic bead and library construction, establish that panonychus citri is micro- to be defended
Star enriched library；However, this method is suitable for developing the microsatellite marker of tiny insect and mite class, and target restrictively
DNA acquisition purity is low, and the reaction susceptibility of PCR amplification is not high.
Summary of the invention
The purpose of the present invention is to provide a kind of construction methods of spot Ji enriched microsatellite library, and develop and can be used for spot Ji
The microsatellite marker of the researchs such as Diversity Detection, Genetic Constitution of Population analysis and paternity identification；This method has simple, high
It imitates, at low cost, Screening SSR Markers accuracy and acquisition are high-efficient, PCR reacts the advantages that success rate is high.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken are as follows:
A kind of construction method of spot Ji enriched microsatellite library, comprising the following steps:
Digestion: extracting genomic DNA from spot Ji muscle or isozyme, using RsaI or MseI to spot Ji genome
DNA carries out digestion；
Connector solution preparation: by connector Spli-A:5 '-GTGACTGCAGGCGTGTGCTCTTCACGA-3 ' and connector
Spli-B:5 '-PO410 μM of aqueous solution is respectively prepared in-GTGACTGGAGTTCCGACGTCTTCACAG-3 ', isometric to mix,
Connector solution is obtained, the connector PCR amplification is high-efficient, does not interfere with each other, and will not generate interference to micro-satellite primers to be amplified；
Connector connection: endonuclease bamhi is mixed with connector solution, to obtain connector-digestion DNA connection product；
Connection product PCR amplification: using connection product as template, PCR reaction is carried out, wherein pcr amplification reaction liquid includes
The primer that 1.5-5 μ LBSA, 1-3.5 μ L concentration is 10 μM, 10-15 μ LPCRMix, 5-8 μ L distilled water, 1-2 μ L connection product should
Under the conditions of, the amplification volume of PCR reaction is few, and Multiple components are pre-mixed, and enormously simplify load procedure, improve library construction
Efficiency；
Enrichment with magnetic bead: the pcr amplification product of connection product is hybridized with biotinylated probes, then by hybrid product and coating
There is the magnetic bead of streptomysin to mix, then elutes the rich segment of magnetic bead surfaces, it is anti-as template progress PCR amplification to elute segment
It answers；
Library construction: the pcr amplification product for eluting segment is connected with cloning vector pMD19-T, is then transferred to large intestine
Bacillus competent cell constructs the clone library of the pico- satellite rich segment of Ji.
Preferably, genomic DNA process is extracted, cyclic DNA and chitin is added, and use Frozen-thawed cycled technical treatment.
It is highly preferred that cyclic DNA is the bacteria plasmid DNA of high-purity；Different size of ring-type can be selected as needed
DNA, as long as added cyclic DNA and constructed target dna library can efficiently separate；The temperature of freezing be -196 to -
20 DEG C, the temperature of thawing is 40-70 DEG C；On the one hand the special presence of cyclic DNA and chitin can promote spot Ji histocyte
Aquation occurs for middle hydrone, thus to the water on spot Ji histocyte protein surface layer in conjunction with nonionic surfactant
Change film and constitute destruction, protein is caused to be easy to assemble to form precipitating, simultaneously because it is normal in the presence of the dielectric that can reduce extracting solution I
Number increases and is extracted attraction in spot Ji histocyte between opposite charges group, promote protein molecule aggregation and
Precipitating, effectively increases the removal efficiency of spot Ji histocyte protein, improves the purity for being extracted tissue DNA；On the other hand,
When being mixed with a large amount of microorganisms (or other heterogeneity biologicals) genome pollution in sample, cyclic DNA and chitin have identification pollution
And the effect of destroying its structure so that extract target dna can uninterruptedly carry out it is subsequent build library, thus microsatellite marker sieves
Make an accurate selection of that exactness is high, it is high-efficient to obtain.
Preferably, endonuclease reaction system are as follows: 1-5 μ LNEB 10 × connection buffer, 0.1-0.5 μ L100 × BSA, 0.1-
0.5 μ L5M NaCl solution, 1-2 μ L restriction enzyme, 10-30 μ L concentration are the genomic DNA of 100ng/ μ L, under this condition,
Inscribe enzyme stability is good, and star activity is low, and the main distribution of DNA fragmentation obtained is 100-1500bp, has reached structure
Build the requirement of spot Ji enriched microsatellite library.
Preferably, chitin fluid is added during configuring pcr amplification reaction liquid, the mass percent of chitin is
0.01%~0.5%, solvent is l-amino acid ethyl ester, and the special presence of chitin fluid has extremely strong adsorption capacity to DNA,
It can be improved PCR success rate, while chitin fluid is made of many microspheres, reaction system can be wrapped up, expand PCR
Increase reaction to complete in separate space small one by one, to improve the reaction susceptibility of PCR amplification, keep reaction more abundant,
Improve the PCR amplification effect of minim DNA；In addition, the boiling point that chitin fluid can improve reaction system is not easy system buffer
Evaporation, reduces the dosage of reagent during PCR amplification, to reduce construction cost.
Preferably, PCR reaction condition are as follows: 90-100 DEG C of denaturation 10s, 55-70 DEG C of annealing 30s, 50-80 DEG C of extension 30s, into
Row 15-20 circulation, last 50-80 DEG C of extension 5min.Under this condition, template DNA unwinding is complete, primer and template renaturation effect
It is good, the efficiency of PCR amplification can be significantly improved.
Preferably, hybridization reaction liquid system during enrichment with magnetic bead are as follows: 10-30 μ L2 × Hyb Solution, 5-15 μ L is raw
The DNA probe TG12 solution of object element label, 5-10 μ L connection DNA fragmentation, 2-8 μ L distilled water.Under this condition, biotin labeling
The complementarity of DNA probe and DNA fragmentation is high, the absorption for being coated with the magnetic bead of streptomysin to hybrid product is improved, to keep away
The waste for having exempted from DNA fragmentation, reduces costs.
Compared with the prior art, the advantages of the present invention are as follows: 1) present invention when extract target gene group DNA be added cyclic annular
DNA and chitin can be improved pollutant in the purity for obtaining DNA, and identification target dna and destroy its structure, to have
It can uninterruptedly carry out subsequent building library conducive to target dna is extracted；2) biotin labeling in the hybridization reaction solution taken
The complementarity of DNA probe and DNA fragmentation is high, improves the absorption for being coated with the magnetic bead of streptomysin to hybrid product；3) in PCR
Chitin fluid is added in amplification reaction solution, PCR amplification effect can be effectively improved, while reducing construction cost.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
A kind of construction method of spot Ji enriched microsatellite library, comprising the following steps:
1) digestion: extracting genomic DNA from spot Ji muscle or isozyme, using restriction enzyme MseI to spot Ji
Genomic DNA carries out digestion, endonuclease reaction system are as follows: 1.5 μ LNEB 10 × connection buffers, 0.1 μ L 100 × BSA, 0.1 μ
L5M NaCl solution, 1 μ L restriction enzyme MseI, 10 μ L concentration are the genomic DNA of 100ng/ μ L；
Extract genomic DNA step are as follows: spot Ji muscle or isozyme sample are shredded, extracting solution I:70ml concentration is placed in
For the Tris-HCl and disodium hydrogen phosphate of 120mM, 0.3g fatty alcohol ether ammonium sulfate, 30ml concentration is the sodium chloride solution of 120mM；
0.5 μ g Plasmid DNA and 0.1 μ g chitin, freeze thawing 2-3 times is added, the temperature of freezing is -126 DEG C, and the temperature of thawing is 45 DEG C,
The Tris-HCl and disodium hydrogen phosphate and 30ml concentration that extracting solution II:10ml concentration is 100mM is added after collecting supernatant in centrifugation
For the cationic polyacrylamide of 0.5ppm, after being mixed by inversion, centrifugation generates white precipitate, and washing precipitates to obtain DNA sample, to
With；On the one hand the special presence of cyclic DNA and chitin can promote hydrone and non-ionic surface in spot Ji histocyte living
Property agent combine, aquation occurs, is destroyed to be constituted to the hydration shell on spot Ji histocyte protein surface layer, causes protein
It is easy to assemble to form precipitating, simultaneously because it has the dielectric constant that can reduce extracting solution I, it is thin that increase is extracted spot Ji tissue
Attraction in born of the same parents between opposite charges group promotes the aggregation and precipitating of protein molecule, effectively increases spot Ji tissue
The removal efficiency of cell protein improves the purity for being extracted tissue DNA；On the other hand, when being mixed with a large amount of microorganisms in sample
(or other heterogeneity biologicals) genome pollution, cyclic DNA and chitin have effects that identification pollutes and destroys its structure, so that
Extract target dna can uninterruptedly carry out it is subsequent build library, thus Screening SSR Markers accuracy is high, it is high-efficient to obtain；
2) prepared by connector solution: by connector the Spli-A:5 '-GTGACTGCAGGCGTGTGCTCTTCACGA-3 ' of synthesis and
Connector Spli-B:5 '-PO410 μM of aqueous solution is respectively prepared in-GTGACTGGAGTTCCGACGTCTTCACAG-3 ', then respectively takes
50 μ L mixing, is added 2 μ L5M NaCl solutions, 95 DEG C of reaction 5min；It is slowly dropped to room temperature, 4 DEG C of standing 3h, -20 DEG C of freezings later
It saves；
3) connector connects: endonuclease bamhi being uniformly mixed with the connector prepared, 16 DEG C are incubated overnight 8 hours or more and carry out
Connection, wherein connection reaction solution system are as follows: 7 μ L joint sequence solution, 1 μ L10 × connection buffer, 2 μ LDNA ligases, 20 μ L
Digestion DNA product；
4) connection product PCR amplification: using connection product as template, to be to draw with the sequence of connector Spli-A sequence complementation
Object carries out PCR reaction, wherein PCR reaction system are as follows: 1.5 μ LBSA, the primer that 1.3 μ L concentration are 10 μM, 10 μ LPCRMix, 5 μ
L distilled water, the chitin fluid that 5 μ L mass concentrations are 0.3%, 2 μ L connection products；PCR reaction condition are as follows: 90 DEG C of denaturation 10s,
60 DEG C of annealing 30s, 55 DEG C of extension 30s carry out 16 circulations, last 55 DEG C of extensions 5min；The special presence of chitin fluid, it is right
DNA has extremely strong adsorption capacity, can be improved PCR success rate, while chitin fluid is made of many microspheres, can be with
Encapsulation reaction system completes pcr amplification reaction in separate space small one by one, so that the reaction for improving PCR amplification is quick
Sensitivity keeps reaction more abundant, improves the PCR amplification effect of minim DNA；In addition, chitin fluid can improve reaction system
Boiling point is not easy to evaporate system buffer, reduces the dosage of reagent during PCR amplification, to reduce construction cost.
5) enrichment with magnetic bead includes the following steps:
A1. the pcr amplification product of connection product hybridizes with biotinylated probes: first by the biotinylated probe of synthesis
(this research probe used is (TG) 12) wiring solution-forming, hybridization reaction liquid system are as follows: 25 μ L2 × Hyb Solution, 10 μ L
12 solution of DNA probe (TG) of biotin labeling, 10 μ L connection DNA fragmentations, 5 μ L distilled waters；Hybridization reaction condition are as follows: first 95
DEG C continue 5min, be then rapidly decreased to 70 DEG C, behind each circulation (5s) successively reduce by 0.2 DEG C, until 50 DEG C, totally 100 are followed
Ring then continues 10min for 50 DEG C, and then each every 5s of circulation successively lowers 0.5 DEG C, and until 40 DEG C, totally 20 are recycled；
A2. magnetic bead pre-processes: 250 μ L TE solution and the 50 coated magnetic beads of μ L Streptavidin are added to 1.5ml centrifugation
Guan Zhong, the 2min that turns upside down clean magnetic bead, and centrifuge tube is then placed on about 1min on Beads enrichment frame, supernatant is sucked, weight
Multiple above-mentioned steps: TE solution 1 time, 1 × Hyb Solution is placed in 150 2 × Hyb of μ L twice, by cleaned magnetic bead
In Solution；
A3. enrichment with magnetic bead: hybrid product is added by mixing, being placed at room temperature for 1h, during which in pretreated magnetic bead solution
Gentle inversion centrifuge tube makes magnetic bead even suspension；
A4. magnetic bead washs: washing carries out in two stages: be slackness washing first: the uniform magnetic bead liquid that will suspend is put
It is placed on magnetic separation rack, stands 1min, supernatant is carefully absorbed, 400 μ L eluent A (2 × SSC 0.1% are then added
SDS), gentle inversion mixes 5min, repeats the above steps 3 times (can be counted with counter), then carries out stringency washes；Then
Be stringency washes: 400 μ L preciseness eluent B (1 × SSC 0.1%SDS) be added, gentle inversion mixes 5min, after by magnetic
Pearl liquid is placed on magnetic separation rack, stands 1min, supernatant is carefully absorbed, is repeated the above steps 3 times；
A5. rich segment elutes: eluting for the first time: 200 μ LTLE buffers being added into the magnetic bead washed, mix.95
DEG C heating water bath 5min is rapidly separated out supernatant with magnetic separation rack, careful Aspirate supernatant, be transferred to another 1.5mL from
In heart pipe, which is first time eluent；Second of elution: the NaOH that 12 μ L 0.15mmol/L are added into magnetic bead is molten
Liquid mixes, and places 20min, during which softly mixes, and is rapidly separated out supernatant with magnetic separation rack later, transfer supernatant is extremely
In another 1.5ml centrifuge tube, the HCl solution and 36.2 μ L TE buffers of 1.8 μ L 1mmol/L are then added in supernatant,
Volume is supplied for 50 μ L, which is second of eluent；
A6. elute fragment amplification: using eluted product twice as substrate, acomplementary connector sequence Spli-A is primer, PCR amplification
System are as follows: 2.5 μ LBSA, 1.3 μ L concentration, which are that 10 μM of acomplementary connector sequence Spli-A, 12.5 μ L PCRMix, 6.7 μ L is bis-, steams
Water, the chitin fluid that 6 μ L mass concentrations are 0.3%, 2 μ L eluted dna segments；PCR response procedures are as follows: 98 DEG C of denaturation 10s, 65
DEG C annealing 30s, 72 DEG C of extensions 30s, carry out 18 recycle, last 72 DEG C of extensions 5min；
6) it secondary enrichment: from agarose gel electrophoresis results, chooses amplification brightness and clip size suitably expands production
Object repeats the above steps as template, carries out second and is enriched with, to improve the bioaccumulation efficiency of microsatellite segment；
7) it elutes fragment amplification product purification: using DNA purification kit (Takara MiniBEST), referring to the reagent
Box operation instructions are operated, and are purified to obtained elution fragment amplification product is enriched with for the second time, the product of acquisition
It is placed in 4 DEG C of preservations；
8) library construction: the pcr amplification product for eluting segment is connected with cloning vector pMD19-T, is then transferred to big
Enterobacteria competent cell constructs the clone library of spot Ji microsatellite rich segment.
Embodiment 2:
A kind of construction method of spot Ji enriched microsatellite library, comprising the following steps:
1) digestion: extracting genomic DNA from spot Ji muscle or isozyme, using restriction enzyme MseI to spot Ji
Genomic DNA carries out digestion, endonuclease reaction system are as follows: 2.5 μ LNEB 10 × connection buffers, 0.25 μ L 100 × BSA, 0.25
μ L5M NaCl solution, 1 μ L restriction enzyme MseI, 20 μ L concentration are the genomic DNA of 100ng/ μ L；
Extract genomic DNA step are as follows: spot Ji muscle or isozyme sample are shredded, extracting solution I:70ml concentration is placed in
For the Tris-HCl and disodium hydrogen phosphate of 120mM, 0.3g fatty alcohol ether ammonium sulfate, 30ml concentration is the sodium chloride solution of 120mM；
0.5 μ g Plasmid DNA and 0.1 μ g chitin, freeze thawing 2-3 times is added, the temperature of freezing is -166 DEG C, and the temperature of thawing is 50 DEG C,
The Tris-HCl and disodium hydrogen phosphate and 30ml concentration that extracting solution II:10ml concentration is 100mM is added after collecting supernatant in centrifugation
For the cationic polyacrylamide of 0.5ppm, after being mixed by inversion, centrifugation generates white precipitate, and washing precipitates to obtain DNA sample, to
With；
2) prepared by connector solution: by connector the Spli-A:5 '-GTGACTGCAGGCGTGTGCTCTTCACGA-3 ' of synthesis and
Connector Spli-B:5 '-PO410 μM of aqueous solution is respectively prepared in-GTGACTGGAGTTCCGACGTCTTCACAG-3 ', then respectively takes
50 μ L mixing, is added 2 μ L5M NaCl solutions, 95 DEG C of reaction 5min；It is slowly dropped to room temperature, 4 DEG C of standing 3h, -20 DEG C of freezings later
It saves；
3) connector connects: endonuclease bamhi being uniformly mixed with the connector prepared, 16 DEG C are incubated overnight 8 hours or more and carry out
Connection, wherein connection reaction solution system are as follows: 7 μ L joint sequence solution, 1 μ L10 × connection buffer, 2 μ LDNA ligases, 20 μ L
Digestion DNA product；
4) connection product PCR amplification: using connection product as template, to be to draw with the sequence of connector Spli-A sequence complementation
Object carries out PCR reaction, wherein PCR reaction system are as follows: 2.5 μ LBSA, the primer that 1.3 μ L concentration are 10 μM, and 12.5 μ LPCRMix,
6.7 μ L distilled waters, the chitin fluid that 6 μ L mass concentrations are 0.3%, 2 μ L connection products；PCR reaction condition are as follows: 98 DEG C of denaturation
10s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 18 circulations, last 72 DEG C of extensions 5min；
5) enrichment with magnetic bead: the pcr amplification product of connection product is hybridized with biotinylated probes, then by hybrid product and packet
There is the magnetic bead of streptomysin to mix, then elutes the rich segment of magnetic bead surfaces, using eluted product as substrate, acomplementary connector sequence
Spli-A is primer, PCR amplification system are as follows: 2.5 μ LBSA, the acomplementary connector sequence SuperSNX-24 that 1.3 μ L concentration are 10 μM,
12.5 μ LPCRMix, 6.7 μ L distilled waters, the chitin fluid that 6 μ L mass concentrations are 0.3%, 2 μ L eluted dna segments；PCR is anti-
Answer program are as follows: 98 DEG C of denaturation 10s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s carry out 18 circulations, last 72 DEG C of extensions 5min；
PCR after reaction, using 1% agarose gel electrophoresis detects yield product length distribution；
6) it secondary enrichment: from agarose gel electrophoresis results, chooses amplification brightness and clip size suitably expands production
Object repeats the above steps as template, carries out second and is enriched with, to improve the bioaccumulation efficiency of microsatellite segment；
7) it elutes fragment amplification product purification: using DNA purification kit (Takara MiniBEST), referring to the reagent
Box operation instructions are operated, and are purified to obtained elution fragment amplification product is enriched with for the second time, the product of acquisition
It is placed in 4 DEG C of preservations；
8) library construction: the pcr amplification product for eluting segment is connected with cloning vector pMD19-T, is then transferred to big
Enterobacteria competent cell constructs the clone library of spot Ji microsatellite rich segment.
Comparative example 1:
It extracts genomic DNA and is added without cyclic DNA and chitin in the process, rest part and embodiment 2 are completely the same；Base
It is lower in the spot Ji microsatellite marker density that this process obtains, illustrate that cyclic DNA and crust mass-energy, which is added, to be effectively improved and extract DNA
Integrity degree, improve spot Ji Screening SSR Markers density.
Comparative example 2:
Chitin fluid is added without in pcr amplification reaction liquid, rest part and embodiment 2 are completely the same；Based on this process
PCR reaction success rate reduce, show chitin fluid it is special exist can be improved the reaction susceptibility of PCR amplification.
Routine operation in operation of the present invention step is well known to those skilled in the art, herein without repeating.
Technical solution of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in spirit of the invention,
Supplement or similar fashion substitution etc., should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of construction method of spot Ji enriched microsatellite library, including digestion, the preparation of connector solution, connector connection, connection production
Object PCR amplification, enrichment with magnetic bead and library construction, it is characterised in that: cyclic DNA is added during extracting spot Ji genomic DNA
And chitin, and extracted using Frozen-thawed cycled processing technique.
2. a kind of construction method of spot Ji enriched microsatellite library according to claim 1, it is characterised in that: the ring-type
DNA is the bacteria plasmid DNA of high-purity.
3. a kind of construction method of spot Ji enriched microsatellite library according to claim 1, it is characterised in that: the freeze thawing
The cryogenic temperature of circular treatment technology is -196 to -20 DEG C, and melt temperature is 40-70 DEG C.
4. a kind of construction method of spot Ji enriched microsatellite library according to claim 1, it is characterised in that: described specific
Step are as follows:
1) digestion: extracting genomic DNA from spot Ji muscle or isozyme, using RsaI or MseI to spot Ji genomic DNA
Carry out digestion；
2) prepared by connector solution: by connector Spli-A:5 '-GTGACTGCAGGCGTGTGCTCTTCACGA-3 ' and connector Spli-
B:5 '-PO410 μM of aqueous solution is respectively prepared in-GTGACTGGAGTTCCGACGTCTTCACAG-3 ', isometric to mix, and obtains connector
Solution；
3) connector connects: endonuclease bamhi being mixed with connector solution, to obtain connector-digestion DNA connection product；
4) connection product PCR amplification: using connection product as template, carrying out PCR reaction, and wherein pcr amplification reaction liquid includes 1.5-5
The primer that μ LBSA, 1-3.5 μ L concentration is 10 μM, 10-15 μ LPCRMix, 5-8 μ L distilled water, 1-2 μ L connection product；
5) enrichment with magnetic bead: the pcr amplification product of connection product is hybridized with biotinylated probes, then by hybrid product be coated with
The magnetic bead of streptomysin mixes, and then elutes the rich segment of magnetic bead surfaces, carries out pcr amplification reaction as template to elute segment；
6) library construction: the pcr amplification product for eluting segment is connected with cloning vector pMD19-T, is then transferred to large intestine bar
Bacterium competence cell constructs the clone library of the pico- satellite rich segment of Ji.
5. a kind of construction method of spot Ji enriched microsatellite library according to claim 4, it is characterised in that: the digestion
Reaction system are as follows: 1-5 μ LNEB 10 × connection buffer, 0.1-0.5 μ L100 × BSA, 0.1-0.5 μ L5M NaCl solution, 1-2
μ L restriction enzyme, 10-30 μ L concentration are the genomic DNA of 100ng/ μ L.
6. a kind of construction method of spot Ji enriched microsatellite library according to claim 4, it is characterised in that: described to match
It sets pcr amplification reaction liquid and chitin fluid is added in the process, the mass percent of chitin is 0.01-0.5%, and solvent is L- ammonia
Base acetoacetic ester.
7. a kind of construction method of spot Ji enriched microsatellite library according to claim 4, it is characterised in that: the PCR
Reaction condition are as follows: 90-100 DEG C of denaturation 10s, 55-70 DEG C of annealing 30s, 50-80 DEG C of extension 30s carry out 15-20 circulation, finally
50-80 DEG C of extension 5min.
8. a kind of construction method of spot Ji enriched microsatellite library according to claim 4, it is characterised in that: the magnetic bead
Hybridization reaction liquid system in enrichment process are as follows: the DNA probe of 10-30 μ L2 × Hyb Solution, 5-15 μ L biotin labeling
TG12 solution, 5-10 μ L connection DNA fragmentation, 2-8 μ L distilled water.
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