CN104212879B - A kind of method based on magnesphere high flux exploitation genome SSR marker - Google Patents
A kind of method based on magnesphere high flux exploitation genome SSR marker Download PDFInfo
- Publication number
- CN104212879B CN104212879B CN201310222359.9A CN201310222359A CN104212879B CN 104212879 B CN104212879 B CN 104212879B CN 201310222359 A CN201310222359 A CN 201310222359A CN 104212879 B CN104212879 B CN 104212879B
- Authority
- CN
- China
- Prior art keywords
- sequence
- ssr
- dna
- library
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 85
- 239000003550 marker Substances 0.000 title claims abstract description 23
- 230000004907 flux Effects 0.000 title claims abstract description 15
- 239000000523 sample Substances 0.000 claims abstract description 53
- 238000013467 fragmentation Methods 0.000 claims abstract description 49
- 238000006062 fragmentation reaction Methods 0.000 claims abstract description 49
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 36
- 239000012634 fragment Substances 0.000 claims abstract description 32
- 238000012408 PCR amplification Methods 0.000 claims abstract description 28
- 238000000746 purification Methods 0.000 claims abstract description 25
- 229960002685 biotin Drugs 0.000 claims abstract description 18
- 235000020958 biotin Nutrition 0.000 claims abstract description 18
- 239000011616 biotin Substances 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 9
- 239000000839 emulsion Substances 0.000 claims abstract description 9
- 239000011324 bead Substances 0.000 claims description 38
- 238000002156 mixing Methods 0.000 claims description 19
- 238000013461 design Methods 0.000 claims description 18
- 239000000047 product Substances 0.000 claims description 17
- 230000007850 degeneration Effects 0.000 claims description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 13
- 238000002372 labelling Methods 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 230000004087 circulation Effects 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- 238000004140 cleaning Methods 0.000 claims description 6
- 239000012149 elution buffer Substances 0.000 claims description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 5
- 238000005352 clarification Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000003643 water by type Substances 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 2
- 229910001873 dinitrogen Inorganic materials 0.000 claims 1
- 238000012163 sequencing technique Methods 0.000 abstract description 21
- 238000009396 hybridization Methods 0.000 abstract description 12
- 238000010276 construction Methods 0.000 abstract description 9
- 230000003321 amplification Effects 0.000 abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 6
- 239000000284 extract Substances 0.000 abstract description 2
- 108091092878 Microsatellite Proteins 0.000 description 118
- 108020004414 DNA Proteins 0.000 description 58
- 241000894007 species Species 0.000 description 17
- 238000002360 preparation method Methods 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 230000003252 repetitive effect Effects 0.000 description 8
- 238000007796 conventional method Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 241000269912 Pseudopleuronectes Species 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 241000388324 Hymenolaimus malacorhynchos Species 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000013600 plasmid vector Substances 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 102100040870 Glycine amidinotransferase, mitochondrial Human genes 0.000 description 2
- 101000893303 Homo sapiens Glycine amidinotransferase, mitochondrial Proteins 0.000 description 2
- 241001468926 Limanda aspera Species 0.000 description 2
- 241001523405 Limax Species 0.000 description 2
- 241000159977 Maritrema novaezealandense Species 0.000 description 2
- 241000470582 Motuweta isolata Species 0.000 description 2
- 241000533524 Powelliphanta augusta Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 238000010926 purge Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000219146 Gossypium Species 0.000 description 1
- 244000299507 Gossypium hirsutum Species 0.000 description 1
- 235000009432 Gossypium hirsutum Nutrition 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 101100476979 Rhodobacter capsulatus sdsA gene Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108020004487 Satellite DNA Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 229940048084 pyrophosphate Drugs 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention discloses a kind of method based on magnesphere high flux exploitation genome SSR marker, comprise the following steps: that (1) extracts target gene group DNA；(2) by gained genomic DNA random fragmentation；(3) genome random fragmentation DNA library is built；(4) DNA library is expanded；(5) biotin labeled SSR probe and the purpose fragment hybridization in library are utilized；(6) the DNA library fragment containing SSR sequence is carried out Streptavidin MagneSphere enrichment；(7) DNA library fragment amplification the purification to enrichment；(8) DNA fragmentation of purification is carried out emulsion-based PCR amplification and order-checking；(9) search sequence containing SSR site in the sequence of gained.This method reduce library construction difficulty, shorten experimental period, improve sequencing throughput, reduce order-checking cost, extensively can apply as the effective ways of the high flux each species SSR marker of exploitation.
Description
Technical field
The invention belongs to biological technical field, develop genome SSR particularly to one based on magnesphere high flux
The method of labelling.
Background technology
SSR (Simple Sequence Repeat), is again microsatellite DNA (Microsatellite DNA).Repeat single
Unit is the shortest, only 1～6bp；Repeat number is variable, and total length is tens bp, is distributed on the diverse location of whole genome.Micro-
The sequence at satellite DNA two ends is usually relatively conservative single-copy sequence, can design special primer accordingly and carry out PCR expansion
Increase.Different according to tandem sequence repeats number, just can reveal that the polymorphism of microsatellite DNA length, the fragment of tool length polymorphism can be used
Make molecular marker.Microsatellite marker is widely used in genetic map structure because of the tool advantage such as rich polymorphism, reproducible, codominance
Build, population genetics, fingerprint analysis, the research field such as phylogeny.
Although SSR marker has many advantages, but the key point affecting SSR use is the exploitation of SSR marker.Only
Develop a certain amount of polymorphism SSR marker, a certain species could be carried out the analysis of SSR marker.Particularly to without reference to
The species (i.e. the genome sequence of these species is not by genome sequencing) of genome sequence, the development and utilization of SSR marker
The most difficult.
The method of SSR marker exploitation has a lot, and traditional method before mainly has: (1) is from public database or relevant literary composition
Offer middle lookup microsatellite locus；(2) PCR primer with reference to nearly edge species SSR site carries out intersection amplification；(3) from genomic DNA
(gDNA), cDNA, EST screen microsatellite locus.First method is severely limited to the data volume in data base, is unsuitable for
The species of unknown gene group sequence；Second method effect is unstable, has higher risk and limitation；The third method
Multiple strategy can be divided into again, sieve storehouse method including classical way, concentration method and omission.For little species that check order, substantially only
The third method can be selected.
In the traditional method screening microsatellite locus from gDNA, cDNA, EST, concentration method is based on SSR probe and gene
The solution hybridization of group DNA library, improves separation efficiency, is a kind of method that Large-scale Screening microsatellite locus is more conventional.
Magnesphere based on first generation order-checking platform needs to divide through genomic library construction, the microsatellite locus rich in microsatellite
Expanding in advance from the order-checking of, positive colony, PCR primer design, primer and the series of steps such as polymorphic detection, flow process is loaded down with trivial details, cost
Height, flux is the most not enough.
High throughput sequencing technologies is a fast-developing in recent years DNA sequence analysis technology, and this technology is relative to first
For sequencing technologies, maximum advantage is that flux is high, and relative cost is low, and speed is fast.The wherein reading of Roche GS FLX+ order-checking platform
The longest, relatively it is suitable for the specific primer design in SSR marker exploitation.This platform is surveyed based on Manganic pyrophosphate complex initiation principle, pyrophosphoric acid
Sequence technology is to be cascaded chemiluminescence reaction by the enzyme in 4 kinds of enzymatic same reaction systems, takes turns in sequencing reaction each, only
Adding a kind of dNTP, if this dNTP matches with template, polymerase just can be incorporated in primer strand and discharge equimolar
The pyrophosphoric acid group (PPi) of number.PPi can be eventually converted into visible light signal, and generates a peak value by optical system.Each
The height of peak value according to being directly proportional to the few nucleotide mixed in reaction, is subsequently adding lower a kind of dNTP, continues the synthesis of DNA.
The application of high throughput sequencing technologies is a main trend of SSR marker exploitation, and application mode includes: (1) directly base
Because of group order-checking；(2) transcript profile order-checking.Directly gene order-checking can obtain substantial amounts of useless sequence, particularly to large-scale or
For complicated genome, cost is high, and efficiency is low；Transcript profile checks order and is only capable of obtaining the SSR marker information of coding region, and coding region
SSR type often have three and the multiple Preference of hexabasic basic weight.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that the method flow for current developing SSR labelling is loaded down with trivial details, cost
Height, inefficient defect, it is provided that a kind of method based on magnesphere high flux exploitation genome SSR marker.
For solving above-mentioned technical problem, one of technical scheme that the present invention takes is: a kind of based on magnesphere high pass
The method of amount exploitation genome SSR marker, described method comprises the following steps:
(1) target gene group DNA is extracted；
(2) by step (1) gained genomic DNA random fragmentation；
(3) genome random fragmentation DNA library is built；
(4) with step (3) gained DNA library as template, utilize before primer, its sequence as shown in SEQ ID NO:1, and after
Primer, its sequence, as shown in SEQ ID NO:2, carries out PCR amplification, it is thus achieved that amplified library fragment；
(5) the purpose fragment containing SSR site is utilized in biotin labeled SSR probe and library to hybridize；
(6) the Streptavidin coated magnetic bead purpose sheet to combining biotin labeled SSR probe in library is utilized
Duan Jinhang is enriched with；
(7) with the purpose fragment of step (6) gained as template, primer before utilizing, its sequence as shown in SEQ ID NO:1,
With rear primer, its sequence, as shown in SEQ ID NO:2, carries out PCR amplification, the DNA fragmentation of purification PCR amplification gained；
(8) it is that template carries out emulsion-based PCR amplification with the DNA fragmentation of the purification of step (7) gained, emulsion-based PCR is expanded institute
Obtain sequence to check order；
(9) in the sequence of step (8) gained, search contains SSR site sequence.
Wherein step (1) is for extracting target gene group DNA.The method of described target gene group DNA extraction is traditional extraction
Method.Various commercial reagent box or other routine techniquess is preferably utilized to extract target gene group DNA.Described target gene group
DNA is preferably Animal genome DNA, plant genome DNA or microbe genome DNA；Described target gene group DNA includes
There is the genomic DNA with reference to genome sequence, or not there is the genomic DNA with reference to genome sequence.
Wherein said is this area conventional conception with reference to genome, refers to that the genome sequence of species is surveyed by full-length genome
Sequence.There is species gene group DNA with reference to genome sequence and refer to that these species are checked order by full genome；Do not have with reference to gene
The genomic DNA of group sequence refers to that these species are not checked order by full genome.
Wherein step (2) is by step (1) gained genomic DNA random fragmentation.Described genomic DNA random fragmentation
Method be this area conventional method, preferably high pressure nitrogen method or utilize fragmentation instrument to carry out fragmentation.Wherein said
Fragmentation instrument preferably: the fragmentation instruments such as Bioruptor Plus or Covaris, described fragmentation instrument is the most commercially available
Can obtain.
Described high pressure nitrogen method is preferably comprised following steps: take the genomic DNA (100 μ l gDNA) of about 4 μ g, 600
Fragmentation system (the gDNA:100 μ l of μ l；Fragmentation buffer: 500 μ l, the component of fragmentation buffer is: 50% glycerol, 5mM
Tris-HCl, 0.5mM EDTA, pH8.0) in, apply the high pressure nitrogen of 0.3～0.4MPa, continue 50～70s.
Described fragmentation instrument is preferably: Bioruptor Plus random fragmentation instrument, described random fragmentation instrument
The parameter of device arranges preferably: Cycle conditions (On/Off times in sec.): 30/90, period: 3～
6cycles.Random fragmentation instrument is preferably: Covaris M220microTUBE AFA Fiber Screw-Cap；This instrument
Device parameter arranges preferably: Peak Incident Power (W): 50, Duty Factor:20%, Cycles per
Burst:200, Treatment Time (s): 25～50s, Temperature (DEG C): 20, Sample volume (μ l): 50.
Random fragmentation the most also includes after terminating being purified fragmentation products, and described purification step is preferably
Utilize various commercial reagent box that gained fragmentation products is purified.Described Kit is preferably Axygen AxyPrepTM
PCR Clean-up Kit.The concrete grammar utilizing described kits fragment see the operation instructions of this test kit.
The fragmentation products of all purification is carried out 1.5%(mass volume ratio) agarose gel electrophoresis, 120V, continue 20min, cut
The fragment of 400～700bp scopes, uses Axygen AxyPrep DNA Gel Extraction Kit to carry out purpose fragment
Glue reclaims purification, uses the eluent of 20～25 μ l to carry out eluting, and concrete grammar see the operation instructions of this test kit.
Wherein step (3) is for building genome random fragmentation DNA library.Described structure genome random fragmentation DNA
The method in library is conventional method.Commercial reagent box is preferably utilized to carry out library construction.Described construction method is preferably
Use Roche GS Rapid Library Prep Kit(test kit) utilize the fragmentation products of above-mentioned purification gained to carry out base
Because group random fragmentation DNA library builds, concrete grammar see the service manual that this test kit of Roche Holding Ag is up-to-date: " Rapid
Library Preparation Method Manual_XL+_May2011》。
Wherein step (4) is as template with step (3) gained DNA library, primer before utilizing, its sequence such as SEQ ID NO:
Shown in 1, and rear primer, its sequence, as shown in SEQ ID NO:2, carries out PCR amplification, it is thus achieved that amplified library fragment.Described PCR expands
The reaction system increased is preferably: genome dna library: 8～15ng；10 × PCR buffer: 5 μ l；DNTP(2.5mM ea.):
4μl；Front primer (10 μMs): 2 μ l；Rear primer (10 μMs): 2 μ l；Archaeal dna polymerase: 0.5 μ l；Add water to 50 μ l.
Wherein said front primer is LibL-Primer-A:
5 '-CCATCTCATCCCTGCGTGTCTCCGACGACT-3 ', its sequence is as shown in SEQ ID NO:1 in sequence table；
Wherein said rear primer is LibL-Primer-B:5 '-CCTATCCCCTGTGTGCCTTG-3 ', its sequence such as sequence
In table shown in SEQ ID NO:2.
The preparation method of described front primer and rear primer is that this area conventional method, preferably complete sequence synthesize and get final product.
Wherein pcr amplification reaction program is preferably: open heat lid to 100 DEG C；(1) 94 DEG C of degeneration 4min；(2) 94 DEG C of changes
Property 30s, (3) 55～62 DEG C annealing 45s, (4) 72 DEG C extend 1min, step (2)～(4) carry out 12～20 circulations；(5) 72 DEG C
Extend 8min.After PCR amplification terminates, the most also include: take 3 μ l amplified productions and carry out 1%(mass volume ratio) agarose gel
Electrophoresis, EB dyes, and detects products distribution scope.
Wherein step (5) is to utilize in biotin labeled SSR probe and library the purpose fragment containing SSR fragment to carry out
Hybridization.Wherein said SSR probe is typical probe, and described SSR probe is preferably 5 ' terminal modified has biotin labeled SSR to visit
Pin.When the genes of interest group of detection is Pseudopleuronectes (Limanda aspera) genome, described biotin labeled SSR probe
Preferably 5 ' the terminal modified mixture having biotin labeling and the following probe of sequence, described sequence is respectively in sequence table
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ
Shown in ID NO:9, i.e. it is respectively (AC) 12, (AG) 12, (AAT) 8, (AGG) 8, (AGC) 8, (AGAT) 6 and (ACAG) 6.
Described SSR probe is preferably 5 ' the terminal modified mixture having biotin labeling and the following probe of sequence, described
Sequence be respectively SEQ ID NO:10 in sequence table, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
Shown in ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, be i.e. respectively (AG) 10, (AC) 10,
(AAC) 8, (AGG) 8, (ACG) 8, (AAG) 8, (ACAT) 6 and (ATCT) 6.Described probe combinations take into account in each species taxonomy
Compatibility between abundance situation and each SSR probe of each SSR site type, and relatively optimize in enrichment with magnetic bead system and condition
Under state, this probe combinations performance is more preferably.The preparation method of described SSR probe is this area customary preparation methods, preferably
For the most synthetically prepared.Described SSR probe properly for primates, other mammals, Rodents, Fish, other vertebrates,
The species widely such as arthropod, terrestrial plant and fungus.
Described SSR probe is conventional method with the method for the purpose fragment hybridization in library, the reaction system of described hybridization:
Genome dna library 50 μ l(100～500ng after amplification)；(concentration of SSR probe is 10 μ to the SSR probe of equimolar mixing
M) 10 μ l；20×SSC30μl；10%SDS1 μ l, hybridization system cumulative volume is 100 μ l.Gained crossbred ties up to run in PCR instrument
Hybridization procedures, this hybridization procedures is preferably comprised following steps: opens heat lid and is preheated to 100 DEG C, 95 DEG C of degeneration 10min, so
Rear each circulation declines 2 DEG C, and 1min is hatched in each circulation, is down to 58 DEG C overnight.The formula of 1 × SSC is: sodium citrate 15mM,
NaCl150mM, pH7.0.
Wherein step (6) is for utilizing the coated magnetic bead of Streptavidin to visit combining biotin labeled SSR in library
The purpose fragment of pin is enriched with.The method of described enrichment with magnetic bead is this area conventional method, and described magnetic bead is for being coated with strepto-
The magnetic bead of Avidin.
The method of described enrichment with magnetic bead is preferably comprised following steps:
(1) prepare magnetic bead: whirlpool mixing is coated with the magnetic bead of Streptavidin, draws 100 μ l immediately, use 200 μ l6 ×
SSC with0.1%SDS washs three times, each 1～2min.Wash complete use magnetic frame every time and fix magnetic bead, absorb washing
Liquid is smooth to magnetic bead.Adding 100 μ l6 × SSC with0.1%SDS after having washed, resuspension is standby.
(2) SSR library enrichment: 100 μ l hybrid product are taken out from PCR instrument, adds ready 100 μ l magnetic beads, gently
Mixing is played in featheriness.Room temperature (20～25 DEG C, lower same) is hatched 30min, every 5min and is mixed gently once, prevents magnetic bead from dropping down onto at the bottom of pipe.
(3) SSR library is cleaned: first enrichment system put to magnetic frame, and room temperature stands 2～3min, absorbs supernatant.Use
200 μ l6 × SSC with0.1%SDS washs three times, each incubated at room 10min, and magnetic frame stands 2min.Then use
3 × SSC with0.1%SDS of 200 μ l58 DEG C preheatings washs three times, hatches 15min for each 58 DEG C, and magnetic frame stands 2min.
Then re-use 200 μ l6 × SSC with0.1%SDS to wash three times, each incubated at room 10min, magnetic frame stands 2min.
Finally use 200 μ l ultra-pure waters or 0.1 × TE to wash twice, on magnetic frame, directly stand 2min after mixing, absorb supernatant.
(4) SSR enriched library eluting: add 25 μ l elution buffers (10mM Tris-HCl, pH8.5) in the magnetic bead after cleaning, mixed
Even rear 95 DEG C of degeneration 5min, are placed on 2min on magnetic frame immediately and clarify to solution, and sucking-off supernatant is continued to employ, and is repeated once, by two
Secondary eluent mixes, and obtains the eluted product of about 50 μ l altogether.
Wherein step (7) be the purpose fragment with step (6) gained as template, utilize before primer, its sequence such as SEQ ID
Shown in NO:1, and rear primer, its sequence, as shown in SEQ ID NO:2, carries out PCR amplification, the DNA sheet of purification PCR amplification gained
Section.
The system of wherein said PCR amplification is this area Standard PCR system, and the system of described PCR amplification is preferably comprised:
Genome dna library 10～15ng；10 × PCR buffer 5 μ l；DNTP(2.5mM ea.) 4 μ l；Front primer (10 μMs) 2 μ l；After
Primer (10 μMs) 2 μ l；Archaeal dna polymerase 0.5 μ l；Supplement ultra-pure water to 50 μ l.The sequence such as SEQ ID of wherein said front primer
Shown in NO:1, the sequence of rear primer is as shown in SEQ ID NO:2.
The response procedures of wherein said PCR amplification is preferably: first turns on heat lid and is preheated to 100 DEG C, (1) 94 DEG C of change
Property 4min, (2) 94 DEG C of degeneration 30s, (3) 58 DEG C annealing 45s, (4) 72 DEG C extend 1min, wherein step (2)～(4) carry out 18 altogether
Individual circulation, (5) 72 DEG C extend 8min.
Wherein said PCR primer purification is this area conventional purification method, and described purification preferably utilizes magnetic bead to carry out
Purification.The method of described magnetic beads for purifying is preferably comprised following steps: add the AMPure of 90 μ l mixings in PCR product
XP Beads(is purchased from Beckman Kurt commerce and trade (Chinese) company limited), incubated at room 15min after mixing, magnetic frame stands
Supernatant is absorbed after 5min.Then 200 freshly prepared 80% ethanol purge of μ l is used twice, each incubated at room 30s, magnetic frame
Upper standing, to clarification, absorbs supernatant.Finally rest on 15min on magnetic frame to be dried to magnetic bead, add 25 μ l elution buffers
(10mM Tris-HCl, pH8.5) eluting twice, gained eluent system the most about 50 μ l.
Wherein step (8) be the DNA fragmentation of the purification with step (7) gained be that template carries out emulsion-based PCR amplification, by emulsion
PCR amplification gained sequence checks order.The method of wherein said emulsion-based PCR amplification preferably see the relevant behaviour of Roche company
Make handbook " emPCR Amp-Lib L SV Method Manual_XL+_May2011 ".Wherein said order-checking is that this area is conventional
Sequencing technologies, preferably utilizes various commercial sequenator to check order, and described sequenator is preferably: Roche GS FLX+
Sequenator.
Wherein step (9) is that search contains SSR site sequence in the sequence of step (8) gained.Described contain in search
The method of SSR site sequence is this area conventional method, preferably utilizes various commercial software to search for SSR site, more preferably
For using MISA software to search for SSR site in order-checking fragment, the parameter of this software is preferably: definition (unit_
size,min_repeats):1-102-63-54-55-56-5；interruptions(max_difference_between_2_
SSRs):100。
It is preferred that the method is additionally included in gained contains design specific PCR amplification on the flanking sequence of SSR site sequence
The step of primer.The method for designing of wherein said specific PCR amplimer is conventional method, preferably utilizes commercial software
Design pcr amplification primer thing, more preferably for using Primer3 software to carry out design of primers.When using this software, design parameter is preferable
Ground is: after design of primers region is five bases of front and rear of the previous base of repetitive sequence, amplified production length 100～
Between 400bp, remaining uses default setting.
On the basis of meeting common sense in the field, above-mentioned each optimum condition, can combination in any, obtain each preferable reality of the present invention
Example.
Agents useful for same of the present invention and raw material are the most commercially.
The most progressive effect of the present invention is: the library processed through enrichment with magnetic bead method of the present invention, wherein
Library fragments without repetitive sequence site is greatly reduced, so that the detection efficiency in repetitive sequence site is greatly improved.At this
On the basis of, in conjunction with library construction mode and the sequencing throughput of high-flux sequence platform, significantly reduce library construction difficulty, reduce
Experimental cost, shorten experimental period, simultaneously because sequencing throughput is greatly improved, thus reduces order-checking cost.We
Method has wide range of applications, and can apply as the effective ways of the high flux multiple species SSR site-tag of exploitation.
Accompanying drawing explanation
Fig. 1 is genome dna library amplified production schematic diagram.Wherein " 1 " is sample, and " C " is negative control, and " M " is
Ladder marker, stripe size is followed successively by 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp from top to bottom.
Fig. 2 is SSR enriched library amplified production schematic diagram.Wherein " 1 " is sample, and " C " is negative control, and " M " is
Ladder marker, stripe size be followed successively by from top to bottom 100bp, 200bp, 300bp, 400bp, 500bp, 600bp,
700bp、800bp、900bp、1000bp、1500bp。
Fig. 3 is a kind of GT repetitive sequence.This sequence is perfect type SSR sequence, number of repetition totally 12 times, microsatellite sequence position
Between 315bp～318bp of order-checking fragment.
Fig. 4 is the order-checking peak shape of a kind of GT repetitive sequence.Wherein sequencing sequence overall length is 459bp, and repetitive sequence is positioned at
Between 315bp～338bp, order-checking signal is normal, and sequence quality is good.
Fig. 5 is a kind of GT repetitive sequence and design of primers sequence.In this sequence, microsatellite sequence is positioned at order-checking fragment
Between 315bp～338bp, can design specific PCR amplimer in its both sides, wherein front primer is positioned at 150bp～169bp
Between, rear primer between 428bp～447bp, a length of 298bp of pcr amplification product.
Detailed description of the invention
Further illustrate the present invention below by the mode of embodiment, but the most therefore limit the present invention to described reality
Execute among example scope.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product description selects.
Embodiment 1 one kinds method based on magnesphere high flux exploitation Pseudopleuronectes SSR marker
1, experiment material: Pseudopleuronectes (Limanda aspera) liver tissue sample 4 parts.Taking 3 Pseudopleuronecteses, place after death takes
Liver organization, is stored in ethanol solution, and room temperature is deposited.Wherein aggregate sample 1 part, is the equivalent of three Pseudopleuronecteses individualities
Liver organization mixture, these species are without with reference to genome sequence.
2, extracting genome DNA: the extraction of genomic DNA uses sky root TIANamp Genomic DNA Kit test kit,
Concrete extracting method operates with reference to the operation instructions of this test kit.
3, genomic DNA random fragmentation: concrete grammar comprises the following steps:
(1) utilize high pressure nitrogen that genomic DNA carries out fragmentation: to take the genomic DNA of 4 μ g, add TE buffer extremely
100 μ l, add in fragmentation cup, are subsequently adding fragmentation solution (50% glycerol, 5mM Tris-HCl, the 0.5mM of 500 μ l
EDTA, pH8.0), piping and druming mixing, install fragmentation device.Connect high-pressure nitrogen bottle and fragmentation device, apply 0.35MPa
Pressure, continue 50s.
(2) solution in Hoarding segment gasifying device, uses Axygen AxyPrep PCR Clean-up Kit to fragmentation
Solution carried out column purification, and concrete purification process is with reference to the operation instructions of this test kit.Use the elution two of 20 μ l
Secondary.
(3) fragmentation DNA eluent is carried out 1.5%(mass volume ratio) agarose gel electrophoresis, 120V electrophoresis
20min.Cut the purpose fragment of 400～700bp scopes, use Axygen AxyPrep DNA Gel Extraction Kit
Purpose fragment carrying out glue and reclaims purification, concrete purification process is with reference to the operation instructions of this test kit.Use the eluting of 20 μ l
Liquid eluting.
4, build genome random fragmentation DNA library: draw 16 μ l fragmentation eluents, use Roche GS Rapid
Library Prep Kit carries out genome dna library structure, and concrete library constructing method is with reference to the use hands of this test kit
Volume " Rapid Library Preparation Method Manual_XL+_May2011 ".
5, the amplification of genome dna library and detection, method particularly includes:
(1) preparation PCR system, as shown in table 1:
Table 1PCR system
| Composition | Volume |
| Genome dna library | 10ng |
| 10 × PCR buffer | 5μl |
| DNTP(2.5mM ea.) | 4μl |
| Front primer (10 μMs) | 2μl |
| Rear primer (10 μMs) | 2μl |
| Archaeal dna polymerase | 0.5μl |
| Supplementary ultra-pure water is extremely | 50μl |
Wherein said front primer is LibL-Primer-A:
5 '-CCATCTCATCCCTGCGTGTCTCCGACGACT-3 ', its sequence is as shown in SEQ ID NO:1 in sequence table；
Wherein said rear primer is LibL-Primer-B:5 '-CCTATCCCCTGTGTGCCTTG-3 ', its sequence such as sequence
In table shown in SEQ ID NO:2.
(2) PCR program is run: open heat lid to 100 DEG C；(1) 94 DEG C of degeneration 4min；(2) 94 DEG C of degeneration 30s, (3) 58 DEG C
Annealing 45s, (4) 72 DEG C extend 1min, and step (2)～(4) carry out 18 circulations；(5) 72 DEG C extend 8min.
(3) from the amplified production in library, take 3 μ l carry out 1%(mass volume ratio) agarose gel electrophoresis, EB dyes, inspection
Surveying products distribution scope, result is as shown in Figure 1.
6, DNA library hybridizes with SSR probe, method particularly includes:
(1) first synthesis terminal modified has biotin labeled SSR probe for 7 kind 5 ', described biotin labeled SSR probe
Sequence is respectively SEQ ID NO:3 in sequence table, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:
7, shown in SEQ ID NO:8 and SEQ ID NO:9, it is (AC) 12, (AG) 12, (AAT) 8, (AGG) 8, (AGC) 8, (AGAT) 6
(ACAG) 6(is synthesized by Sani bio tech ltd, Shanghai), it is diluted to 10 μMs etc. after being dissolved by above-mentioned 7 kinds of SSR probes
Volume mixture.
(2) preparation probe hybridization system, as shown in table 2:
Table 2 probe hybridization system
| Composition | Volume |
| Genome dna library | 200ng |
| SSR probe (10 μMs) | 10μl |
| 20×SSC | 30μl |
| 10%SDS | 1μl |
| Supplementary ultra-pure water is extremely | 100μl |
(3) hybridization program is run: open PCR instrument heat lid to 100 DEG C, first 95 DEG C degeneration 10min, the most each follow
Ring declines 2 DEG C, and hatches 1min, drops to 58 DEG C and keeps overnight.
7, SSR library enrichment, cleaning, eluting, method particularly includes:
(1) magnetic bead is prepared: whirlpool mixing is coated with the magnetic bead of Streptavidin (purchased from the Shanghai English limited public affairs of fine horse biotechnology
Department), draw 100 μ l immediately, use 200 μ l6 × SSC with0.1%SDS to wash three times, each 2min.Washing is complete every time makes
Fix magnetic bead with magnetic frame, absorb cleaning mixture, smooth to magnetic bead, add 100 μ l6 × SSC with0.1%SDS after having washed,
Resuspension is standby.
(2) SSR library enrichment: 100 μ l hybrid product are taken out from PCR instrument, adds ready 100 μ l magnetic beads, gently
Mixing is played in featheriness.Room temperature (20～25 DEG C, lower same) is hatched 30min, every 5min and is mixed gently once, prevents magnetic bead from dropping down onto at the bottom of pipe.
(3) SSR library is cleaned: first enrichment system put to magnetic frame, and room temperature stands 2min, absorbs supernatant.Use 200
μ l6 × SSC with0.1%SDS washs three times, each incubated at room 10min, and magnetic frame stands 2min.Then 200 μ are used
3 × SSC with0.1%SDS of l58 DEG C of preheating washs three times, hatches 15min for each 58 DEG C, and magnetic frame stands 2min.Then
Re-use 200 μ l6 × SSC with0.1%SDS to wash three times, each incubated at room 10min, magnetic frame stands 2min.Finally
Use 200 μ l ultra-pure waters or 0.1 × TE to wash twice, on magnetic frame, directly stand 2min after mixing, absorb supernatant.
(4) SSR enriched library eluting: to clean after magnetic bead in add 25 μ l elution buffers (10mM Tris-HCl,
PH8.5), mixing rear 95 DEG C of degeneration 5min, be placed on 2min on magnetic frame immediately and clarify to solution, sucking-off supernatant is continued to employ, and repeats
Once, by twice eluent mixing, the eluted product of about 50 μ l altogether is obtained.
8, the amplification of SSR enriched library and purification, method particularly includes:
(1) preparation PCR reaction system, as shown in table 3:
Table 3PCR reaction system
| Composition | Volume |
| SSR enriched library | 10μl |
| 10 × PCR buffer | 5μl |
| DNTP(2.5mM ea.) | 4μl |
| Front primer (10 μMs) | 2μl |
| Rear primer (10 μMs) | 2μl |
| Archaeal dna polymerase | 0.5μl |
| Supplementary ultra-pure water is extremely | 50μl |
The sequence of wherein said front primer is as shown in SEQ ID NO:1 in sequence table；The sequence of wherein said rear primer is such as
In sequence table shown in SEQ ID NO:2.
(2) PCR response procedures is run: first turn on heat lid and be preheated to 100 DEG C, (1) 94 DEG C of degeneration 4min, (2) 94 DEG C
Degeneration 30s, (3) 58 DEG C of annealing 45s, (4) 72 DEG C extend 1min, and wherein step (2)～(4) carry out 18 circulations altogether, (5) 72 DEG C
Extend 8min.
(3) PCR primer purification: add the AMPure XP Beads(of 90 μ l mixings in PCR product purchased from Bake
Graceful Kurt commerce and trade (Chinese) company limited), incubated at room 15min after mixing, absorb supernatant after magnetic frame stands 5min.So
Rear freshly prepared 80% ethanol purge of use 200 μ l twice, each incubated at room 30s, magnetic frame stands to clarification, absorb
Supernatant.Finally rest on 15min on magnetic frame to be dried to magnetic bead, add 25 μ l elution buffers (10mM Tris-HCl,
PH8.5) eluting twice, altogether about 50 μ l.
(4) from the amplified production in library, take 3 μ l carry out 1%(mass volume ratio) agarose gel electrophoresis, EB dyes, inspection
Surveying products distribution scope, result is as shown in Figure 2.
9, SSR enriched library emulsion-based PCR expands and machine order-checking on Roche GS FLX+ sequenator.Concrete grammar refers to
Roche company associative operation handbook " emPCR Amp-Lib L SV Method Manual_XL+_May2011 " and " GS
FLX+_Sequencing_Method Manual_XL+Kit_May2011》。
10, using MISA software to search for SSR site in sequencing sequence, use parameter is: definition (unit_
size,min_repeats):1-102-63-54-55-56-5；interruptions(max_difference_between_2_
SSRs):100.Sequencing sequence output and SSR site Search Results are as shown in table 4:
Table 4 sequencing sequence output and SSR site Search Results
| Sample | reads | No.SSR | SSR development efficiency |
| 1 | 14,056 | 5,453 | 38.79% |
| 2 | 38,957 | 21,202 | 54.42% |
| 3 | 28,799 | 10,349 | 35.94% |
| 4 | 29,801 | 14,482 | 48.60% |
Segments obtained by wherein " reads " is each sample order-checking, " No.SSR " is the survey that each sample contains SSR site
Sequence segments.Fig. 3 shows the sequencing sequence of a preferable microsatellite locus, and Fig. 4 shows its peak shape figure that checks order.
It can be seen that use the method for the invention can to develop in single experiment much more very from above table
SSR number of sites, and library construction flow process is fast, technical difficulty is low, experimental cost is low, and sequencing data flux is high, SSR detection efficiency
Up to 35～55%, in multiple hereditism application relevant for SSR, to the high flux of SSR marker in the range of each species full-length genome
Exploitation serves huge facilitation.
11, use Primer3 software that the sequence containing SSR site detected is carried out specific primer design, use
Parameter is: after design of primers region is five bases of front and rear of the previous base of repetitive sequence, amplified production length is 100
～between 400bp, remaining uses default setting.The SSR number of sites that can design primer sequence is as shown in table 5:
Table 5 can design the SSR number of sites of primer sequence
Fig. 5 shows sequencing sequence and the design of primers site thereof of one of them preferable microsatellite locus.
It can be seen that the SSR bit number of points using the method for the invention to develop is the most from above table, Er Qiexu
Row length is long, beneficially the design of specific PCR amplimer, and the SSR site ratio that can design primer is up to 40～60%.
Comparative example 1
Blue duck (Hymenolaimus malacorhynchos), trematodiasis is extracted respectively according to method described in embodiment 1
(Maritrema novaezealandensis), weta (Motuweta isolata) and Limax (Powelliphanta
Augusta) genomic DNA of four kinds of samples, and build with Bigpian according to the genomic DNA to extracting of the method described in embodiment 1
Sectionization DNA library, then directly carries out machine in emPCR amplification and Roche GS FLX sequenator according to method described in embodiment 1
Order-checking steps such as (eliminate amplified library therein) the SSR probe hybridization of labelling biotin, enrichment with magnetic bead, cleaning, eluting,
Gained sequencing sequence output and SSR site Search Results are as shown in table 6:
Table 6 sequencing sequence output and SSR site Search Results
| Sample | reads | No.SSR | SSR development efficiency |
| Blue duck (Hymenolaimus malacorhynchos) | 17,215 | 231 | 1.34% |
| Trematodiasis (Maritrema novaezealandensis) | 31,120 | 676 | 2.17% |
| Weta (Motuweta isolata) | 52,059 | 472 | 0.91% |
| Limax (Powelliphanta augusta) | 35,196 | 2,541 | 7.22% |
Above-mentioned four kinds of preparation methoies stating sample, refer to document: Abdelkrim J, Robertson B C, Stanton
J A L,et al.Fast,cost-effective development of species-specific
microsatellite markers by genomic sequencing[J].Biotechniques,2009,46(3):185.
Segments obtained by wherein " reads " is each sample order-checking, " No.SSR " is the survey that each sample contains SSR site
Sequence segments.
From the data of table 6 it can be seen that the method is not owing to having SSR site to genome random fragment library
Nucleic acid fragment is enriched with, and therefore comprises substantial amounts of non-SSR sequence (without the sequence in SSR site), SSR marker in sequencing sequence
Development efficiency is extremely low.
Comparative example 2
The method that comparative example 2 uses is to highland cotton variety (Gossypium hirsutum L) plant young tender leaf
Sheet extracts genomic DNA, uses high pressure nitrogen by the genomic DNA random fragmentation of 10 μ g to 450-900bp scope, then enters
Row ethanol deposition and purification and again dissolve (preparation method of above-mentioned sample, refer to document: Connell J P, Pammi S,
Iqbal M J,et al.A high through-put procedure for capturing microsatellites
from complex plant genomes[J].Plant Molecular Biology Reporter,1998,16(4):
341-349.).Use T4DNA polymerase and Klenow fragment incubated at room 30min by DNA end-filling, re-use T4 and connect
Enzyme 16 DEG C overnight connects double-stranded adapters, builds genome dna library.Then by biotin labeled to 100ngDNA library and 6 kinds
SSR probe is (sequence of described probe respectively: (GA)20、(CA)20、(AGA)15、(ACA)15、(CAT)15(CTA)15) mixing,
95 DEG C of degeneration 5min, are down to 60 DEG C for 2 DEG C per minute, keep 1h, by SSR probe and the library fragments hybridization containing SSR site.Profit
With carrying out SSR hybridized fragment with the Streptavidin MagneSphere of the biotin labeling specific bond on SSR probe and magnetic frame
The steps such as enrichment, cleaning and eluting, specific experiment method asks for an interview embodiment 1.Subsequently use lead to corresponding with double-stranded adapters
PCR amplification is carried out with the primer single stranded DNA to eluting.Select plasmid vector pCRII (Invitrogen) afterwards, use
EcoR I restriction enzyme cleavage, utilizes ligase 16 DEG C overnight that DNA fragmentation is special with two sticky ends of plasmid vector
Property connect, re-use electroporation technology and plasmid vector be transformed in escherichia coli.Preparation YT-agar culture medium (train by YT-agar
The formula supporting base is: peptone 10.0g yeast extract 5.0g, glucose 1.0g, NaCl5.0g, agar 20.0g, distilled water
1000ml, pH7.0), and add ammonia benzyl penicillium sp Amp(0.1% percent by volume), X-GAL(final concentration 40 μ g/ml) and IPTG(is eventually
Concentration 0.5mmol/L), escherichia coli are converted successful white colony in 37 DEG C of incubated overnight, picking.Carry out subculture training again
Supporting, therefrom some white colonies of picking carry out plasmid extraction and purification, and DNA Insert Fragment therein is carried out PCR amplification, utilize
Machine order-checking on generation Sanger order-checking platform ABI377, processes sequencing result and searches for SSR site sequence.Present case
In, build SSR enriched library time-consuming about one week, library is screened and utilizes sequencer then to need one month
Can complete.Testing result shows, after strict screening, in the SSR enriched library developed, SSR site content is more than 20%, altogether
Check order and identify more than 500 SSR sites.
The method has following several big shortcoming compared with the method for the invention: 1) experiment flow is long, complex operation, concrete table
It is now: after SSR enrichment, to carry out the steps such as library construction, positive colony screening and qualification, plasmid extraction and purification of recombinating,
Flow process is loaded down with trivial details, and positive colony detection ratio is low in the case of sample quality is poor, operation complexity；2) response time is longer:
This experiment flow compares the method for the invention at least to have more the time of 3～5 days；3) flux of the method order-checking is relatively low: the
Generation Sanger order-checking platform each run can only detect 96 positive colonies simultaneously, i.e. produces 96 sequencing sequences, and this
Bright based on secondary order-checking platform GS Roche FLX+, once operation can be checked order and be reached million sequences, and sequencing throughput has had greatly
Improve, thus SSR marker detection efficiency is also greatly promoted；4) experimental cost is higher: owing to adding restructuring library construction, plasmid
The steps such as extracting add the cost of about 100 yuan to each clone, the order-checking cost of each clone about 2 yuan.And it is of the present invention
Method improves greatly due to flux, and the sequence cost of each clone is less than 0.2 yuan.Furthermore, it is necessary to special instruction, we
Method has higher species universality, and the Exact Design of different SSR probe type does not affect the method developing SSR labelling with using
High efficiency.
It will further be understood that those skilled in the art do change or the improvement of part in the method flow process, all should fall into
The application appended claims limited range.
Claims (8)
1. a method based on magnesphere high flux exploitation genome SSR marker, it is characterised in that described method includes
The following step:
(1) target gene group DNA is extracted；
(2) by step (1) gained genomic DNA random fragmentation；
(3) genome random fragmentation DNA library is built；
(4) with step (3) gained DNA library as template, utilize before primer, its sequence as shown in SEQ ID NO:1, and after draw
Thing, its sequence, as shown in SEQ ID NO:2, carries out PCR amplification, it is thus achieved that amplified library fragment；
(5) the purpose fragment containing SSR site is utilized in biotin labeled SSR probe and library to hybridize；Described biology
The SSR probe of element labelling is 5 ' the terminal modified mixture having biotin labeling and the following probe of sequence, and described sequence is respectively
For SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID in sequence table
Shown in NO:8 and SEQ ID NO:9；
(6) utilize Streptavidin coated magnetic bead that the purpose fragment combining biotin labeled SSR probe in library is entered
Row enrichment；
(7) with the purpose fragment of step (6) gained as template, primer before utilizing, its sequence as shown in SEQ ID NO:1, and after
Primer, its sequence, as shown in SEQ ID NO:2, carries out PCR amplification, the DNA fragmentation of purification PCR amplification gained；
(8) it is that template carries out emulsion-based PCR amplification with the DNA fragmentation of the purification of step (7) gained, emulsion-based PCR is expanded gained sequence
Row check order；
(9) search sequence containing SSR site in the sequence of step (8) gained.
2. the method for claim 1, it is characterised in that step (1) described target gene group DNA is Animal genome
DNA, plant genome DNA or microbe genome DNA.
3. the method for claim 1, it is characterised in that the method for step (2) described DNA random fragmentation is elevated pressure nitrogen
Gas method, the method for described random fragmentation comprises the following steps: apply the high pressure nitrogen of 0.3～0.4MPa to fragmentation system,
Continue 50～70 seconds.
4. the method for claim 1, it is characterised in that the program of step (4) described PCR amplification is: open heat lid extremely
100℃；(1) 94 DEG C of degeneration 4 minutes；(2) 94 DEG C of degeneration 30 seconds, anneal 45 seconds for (3) 55～62 DEG C, and (4) 72 DEG C extend 1 minute,
Step (2)～(4) carry out 12～20 circulations；(5) 72 DEG C extend 8 minutes.
5. the method for claim 1, it is characterised in that step (6) is described utilizes the coated magnetic bead pair of Streptavidin
Library combines the method that the purpose fragment of biotin labeled SSR probe carries out being enriched with comprise the following steps:
(1) magnetic bead is prepared: whirlpool mixing is coated with the magnetic bead of Streptavidin, draws 100 μ l immediately, uses 200 μ l to include
6 × the SSC of 0.1%SDS washs three times, each 1～2 minute, washs complete use magnetic frame every time and fixes magnetic bead, absorb washing
Liquid, smooth to magnetic bead, add 100 μ l after having washed and include the 6 × SSC of 0.1%SDS, resuspension is standby；
(2) SSR library enrichment: add ready 100 μ l magnetic beads in 100 μ l step (5) gained hybrid product, blow and beat gently
Mixing, hatches 30 minutes for 20～25 DEG C, within every 5 minutes, mixes gently once, prevent magnetic bead from dropping down onto at the bottom of pipe；
(3) SSR library is cleaned: first enrichment system put to magnetic frame, and room temperature stands 2～3 minutes, absorbs supernatant, uses 200
μ l includes the 6 × SSC of 0.1%SDS and washs three times, hatches 10 minutes for each 20～25 DEG C, magnetic frame stands 2 minutes, then
3 × the SSC including 0.1%SDS using 200 μ l 58 DEG C to preheat washs three times, hatches 15 minutes, on magnetic frame for each 58 DEG C
Stand 2 minutes, then re-use 200 μ l include 0.1%SDS 6 × SSC wash three times, hatch 10min for each 20～25 DEG C,
2 minutes are stood on magnetic frame；200 μ l ultra-pure waters or 0.1 × TE is finally used to wash twice, directly quiet on magnetic frame after mixing
Put 2 minutes, absorb supernatant；
(4) SSR enriched library eluting: adding 25 μ l elution buffers in the magnetic bead after cleaning, described elution buffer is dense
Degree is the Tris-HCl of 8.5 for 10mM, pH, mixes rear 95 DEG C of degeneration 5 minutes, is placed on magnetic frame upper 2 minute immediately to solution
Clarification, sucking-off supernatant is continued to employ, and is repeated once, and by twice eluent mixing, obtains the eluted product of totally 50 μ l.
6. the method for claim 1, it is characterised in that the system of the PCR amplification described in step (7) is: combine life
The fragment 10 μ l of the SSR probe of thing element labelling；10 × PCR buffer 5 μ l；dNTP4μl；Front primer 2 μ l；Rear primer 2 μ l；DNA
Polymerase 0.5 μ l；Add water to 50 μ l, described front primer sequence as shown in SEQ ID NO:1, described rear primer sequence such as SEQ ID
Shown in NO:2, the program of described PCR amplification is: (1) 94 DEG C of degeneration 4 minutes, (2) 94 DEG C of degeneration 30 seconds, (3) 55～62 DEG C of annealing
45 seconds, (4) 72 DEG C extended 1 minute, and step (2)～(4) carry out 12～20 circulations；(5) 72 DEG C extend 10 minutes.
7. the method for claim 1, it is characterised in that step (8) described order-checking is to be checked order by Roche GS FLX+
Instrument is carried out.
8. the method for claim 1, it is characterised in that contain the flanking sequence of SSR site sequence in step (9) gained
Upper design specific PCR amplimer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310222359.9A CN104212879B (en) | 2013-06-05 | 2013-06-05 | A kind of method based on magnesphere high flux exploitation genome SSR marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310222359.9A CN104212879B (en) | 2013-06-05 | 2013-06-05 | A kind of method based on magnesphere high flux exploitation genome SSR marker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104212879A CN104212879A (en) | 2014-12-17 |
| CN104212879B true CN104212879B (en) | 2016-09-21 |
Family
ID=52094783
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310222359.9A Active CN104212879B (en) | 2013-06-05 | 2013-06-05 | A kind of method based on magnesphere high flux exploitation genome SSR marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104212879B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106103742B (en) | 2015-01-06 | 2019-12-10 | 深圳市海普洛斯生物科技有限公司 | Method and reagent for enriching circulating tumor DNA |
| CN106987648B (en) * | 2017-06-01 | 2020-07-17 | 中国农业大学 | High-flux SSR molecular marking method related to plant organ development |
| CN107904666A (en) * | 2017-12-15 | 2018-04-13 | 中源协和基因科技有限公司 | A kind of library construction and quantitative approach applied to tumour driving genetic test |
| CN109355365A (en) * | 2018-11-25 | 2019-02-19 | 中国农业科学院兰州兽医研究所 | A kind of method of tiny RNA high-flux sequence detection microorganism |
| CN110241462A (en) * | 2019-06-27 | 2019-09-17 | 深圳市海普洛斯生物科技有限公司 | A kind of method and apparatus of two generations sequencing library targeting fast enriching |
| CN110387404B (en) * | 2019-06-28 | 2020-07-28 | 浙江大学 | Magnetic bead-PNA probe compound and application of magnetic bead-PNA probe compound in enrichment of liver cancer circulating tumor DNA |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1295615A (en) * | 1998-03-27 | 2001-05-16 | 大塚制药株式会社 | Human p51 genes and gene products thereof |
| EP2096173A1 (en) * | 2003-05-21 | 2009-09-02 | Asahi Kasei Pharma Corporation | Method of measuring glycolated hemoglobin A1C, enzyme to be used therefor and process for producing the same |
-
2013
- 2013-06-05 CN CN201310222359.9A patent/CN104212879B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1295615A (en) * | 1998-03-27 | 2001-05-16 | 大塚制药株式会社 | Human p51 genes and gene products thereof |
| EP2096173A1 (en) * | 2003-05-21 | 2009-09-02 | Asahi Kasei Pharma Corporation | Method of measuring glycolated hemoglobin A1C, enzyme to be used therefor and process for producing the same |
Non-Patent Citations (1)
| Title |
|---|
| 条斑紫菜功能基因组及重复序列特征研究;杨惠;《中国博士学位论文全文数据库农业科技辑》;20120215(第2期);摘要，第9页第2.4节，第78页第1段，第79页第1.3节，第82页第2节，图5-1，第83-86页第2.1、2.2节，第86-87页第2.3节，第90页第2.4节，第109页第1节，第110-112页第2节，图6.2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104212879A (en) | 2014-12-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6959378B2 (en) | Enzyme-free and amplification-free sequencing | |
| CN104212879B (en) | A kind of method based on magnesphere high flux exploitation genome SSR marker | |
| US10072283B2 (en) | Direct capture, amplification and sequencing of target DNA using immobilized primers | |
| US11072819B2 (en) | Methods of constructing small RNA libraries and their use for expression profiling of target RNAs | |
| CN106434865B (en) | DNA typing method and kit for HLA gene | |
| EP3006571B1 (en) | Hla gene multiplex dna typing method and kit | |
| US20100035249A1 (en) | Rna sequencing and analysis using solid support | |
| CN108611398A (en) | Genotyping is carried out by new-generation sequencing | |
| WO2023060871A1 (en) | Hla gene amplification primer, kit, sequencing library establishment method, and sequencing method | |
| US20140336058A1 (en) | Method and kit for characterizing rna in a composition | |
| US20060228714A1 (en) | Nucleic acid representations utilizing type IIB restriction endonuclease cleavage products | |
| WO2017024991A1 (en) | Swine bcr heavy-chain multiplex pcr primer and application thereof | |
| WO2011070155A1 (en) | Rna analytics method | |
| Melamed et al. | Exploring translation regulation by global analysis of ribosomal association | |
| Duftner et al. | Efficacy of RNA amplification is dependent on sequence characteristics: implications for gene expression profiling using a cDNA microarray | |
| Breter et al. | Approaches for a sustainable use of the bioactive potential in sponges: analysis of gene clusters, differential display of mRNA and DNA chips | |
| US10036053B2 (en) | Determination of variants produced upon replication or transcription of nucleic acid sequences | |
| CN109355288B (en) | Method for enriching target DNA and application | |
| CN114250279B (en) | Construction method of haplotype | |
| Tinio et al. | Development of microsatellite markers from genomic DNA of Parashorea malaanonan (Dipterocarpaceae) using next-generation sequencing | |
| Wang et al. | Novel 28 microsatellite loci using high-throughput sequencing for an endangered species on Metasequoia glyptostroboides (Cupressaceae) | |
| WO2009079733A1 (en) | Determination of variants produced upon replication or transcription of nucleic acid sequences | |
| CN112064121A (en) | Multiple PCR sequencing method for collecting RNA reverse transcription, enriching and establishing library and library establishing instrument | |
| CN114250279A (en) | Method for constructing haplotype | |
| CN116377084A (en) | High-efficiency autosomal micro-haplotype genetic marker system, and detection primer and kit thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 200231, room 7, building 777, 301 Wu Long Road, Shanghai, Xuhui District Applicant after: SHANGHAI PERSONAL BIOTECHNOLOGY CO., LTD. Address before: 200231, room 7, building 777, 301 Wu Long Road, Shanghai, Xuhui District Applicant before: Shanghai Personal Biotechnology Co., Ltd. |
|
| COR | Change of bibliographic data | ||
| CB02 | Change of applicant information |
Address after: 200231 Shanghai city Xuhui District Yindu Road No. 218, poly Park 2 building Applicant after: SHANGHAI PERSONAL BIOTECHNOLOGY CO., LTD. Address before: 200231, room 7, building 777, 301 Wu Long Road, Shanghai, Xuhui District Applicant before: SHANGHAI PERSONAL BIOTECHNOLOGY CO., LTD. |
|
| COR | Change of bibliographic data | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Magnetic bead enrichment technique based method for high-flux development of genome SSR markers Effective date of registration: 20190328 Granted publication date: 20160921 Pledgee: Anxin Agricultural Insurance Co., Ltd. Shanghai Minhang Branch Company Pledgor: SHANGHAI PERSONAL BIOTECHNOLOGY CO., LTD. Registration number: 2019310000011 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20201019 Granted publication date: 20160921 Pledgee: Anxin Agricultural Insurance Co., Ltd. Shanghai Minhang Branch Co. Pledgor: SHANGHAI PERSONAL BIOTECHNOLOGY Co.,Ltd. Registration number: 2019310000011 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |