CN101705296A - Detection method of turbot FF0922 microsatellite marker by utilizing specific primers - Google Patents
Detection method of turbot FF0922 microsatellite marker by utilizing specific primers Download PDFInfo
- Publication number
- CN101705296A CN101705296A CN200910230906A CN200910230906A CN101705296A CN 101705296 A CN101705296 A CN 101705296A CN 200910230906 A CN200910230906 A CN 200910230906A CN 200910230906 A CN200910230906 A CN 200910230906A CN 101705296 A CN101705296 A CN 101705296A
- Authority
- CN
- China
- Prior art keywords
- turbot
- sequence
- primer
- microsatellite
- pcr
- 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.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a detection method of a turbot FF0922 microsatellite marker by utilizing specific primers, comprising the following steps: firstly extracting turbot genomic DNA; then utilizing a sequence containing a microsatellite in an enrichment library of the turbot microsatellite; designing specific primers at two ends of the sequence as a positive strand 5'-TGTGAGAAAACAGGCAATC-3', and a negative strand CAGTTTCTGTGATTTGGTGAT-3'; carrying out PCR amplification on the turbot genomic DNA; separating the obtained PCR product through modified polyacrylamide gel electrophoresis; analyzing strips generated on the product to obtain a turbot genetic polymorphism map. The method of the invention is suitable for the detection technologies of turbot population genetic marker, genealogy authentication, genetic linkage map construction and the like; the PCR amplification product presents better polymorphism in turbot population detection; and the method of the invention can rapidly acquire a genetic variation map of the turbot FF0922 microsatellite marker, and conveniently, rapidly, accurately and intuitively detect each individual genotype of the turbot.
Description
Technical field:
The invention belongs to turbot dna molecular genetic marker technology, is a kind of detection turbot FF0922 micro-satellite labeling technique method.
Background technology:
Before the present invention makes, similar report is arranged both at home and abroad, Isolation andcharacterization of polymorphic microsatellite loci from ESTlibrary of turbot (Scophthalmus maximus) the and cross-speciesamplification that (Molecular Ecology Notes 2007) such as the old pine forests of domestic Chinese aquatic science Huanghai Sea aquatic products institute of institute delivers, once reported the detection method of turbot part microsatellite locus, but the turbot genomic library is not oneself to be made up, but choose in the existing report; Isolation and characterization of microsatellitemarkers for turbo (Scophthalmus maximus) the by screening SSR-enrichedlibrary that Sun Xiaowen etc. (ConservGenet 2009) deliver, once reported turbot portion gene group library structure, contain the contents such as screening of microsatellite sequence, but the sequence allelotrope that its primer amplification that has goes out very little, polymorphism is not high, thereby can not distinguish different groups or individuality effectively when causing using; The Development and characterization of 248 novel microsatellitemarkers in turbot that deliver (Genome 2007) such as Spain Pa rdo, once report filtered out 248 microsatellite locus with the method that makes up the turbot enriched microsatellite library, and designed primer and provide condition, but do not see report to each primer polymorphism situation for making up genetic linkage maps.
Summary of the invention:
The objective of the invention is to propose a kind of autonomous structure turbot gene enriched library, and therefrom filter out microsatellite sequence, carrying out PCR in its design specific primers at both ends detects, thereby detect of the heritable variation of each individuality of turbot fast in little satellite district, obtain this primer to turbot polymorphism collection of illustrative plates, detect each individual genotype by collection of illustrative plates, in the hope of finding the proterties chain, carry out breeding work and lay the foundation for combining with traditional method later on this microsatellite locus.
The present invention finishes according to following operational aspect: at first be to extract turbot genomic dna and diluted for use; Utilize the little satellite core sequence that contains in the turbot genomic library again, in its design specific primers at both ends; Use this primer that the genomic dna of Different Individual in different geographical populations of turbot or the same colony is carried out pcr amplification then, pcr amplification product is detected; The band that utilizes product to occur is analyzed, and determines the genotype that each is individual, and obtains the genetic polymorphism collection of illustrative plates of turbot.
Extract the turbot genomic dna its dilution is 50ng/ul, add 2ul in each PCR reaction, the reaction cumulative volume is 25ul.
Little satellite core sequence of FF0922 and two ends conserved sequence thereof are:
1 ATGTGCTGTG?TCAGACTGTT?TATCTGTGAG?AAAACAGGCA?ATCAGAGAGG
51 GCGCTCACAT?ATTATTGAAA?TGACATGAAA?TCCATGTTAC?TCCACACTCA
101?AGTCGTATAT?GCAAGCTAGA?ATGGTGAATC?TCTTCTTGTC?TCCTACAGAC
151?AGACAAACAC?ACACACACAC?ACACACACAC?ACACACACAC?ACAAACACAT
201?ACACAAAACA?AGACCAACAG?TGACATGATA?TAACATGATC?ACCAAATCAC
251?AGAAACTGTA?GGGGAGAAGG?GGAAAAAAAG?CTAATAAAGC?TATAGCAAAA
301?TTAGAACTGT?GCTGTAGGCA?GATCCAGTGT?CTCCAGTAAT?CTACATAAGC
351?AGCAGTTGTC?GT；
Auele Specific Primer according to its design is: normal chain 5 '-TGTGAGAAAACAGGCAATC-3 ' minus strand 5 '-CAGTTTCTGTGATTTGGTGAT-3 ', annealing temperature is 60 ℃ when using primer.
The application of sample parameter of pcr amplification is: each PCR reaction cumulative volume is 25ul, comprises 100ng turbot genomic dna; 10 * PCR buffer, 2.5ul; Mg
2+, 2mmol/L; Taq enzyme 1U; Each 0.2mmol/L of dNTP; The present invention is at each 0.2umol/L of primer of the little satellite core sequence of FF0922 two ends design; Add ddH
2O to 25ul.The program that the PCR instrument is set when using this primer is 94 ℃ of sex change 4min; 94 ℃ of 30sec, 60 ℃ of 50sec, 72 ℃ of 50sec, 30 circulations; 72 ℃ are extended 7min, 4 ℃ of preservations.
The product of PCR detects: is 8% denaturing polyacrylamide gel with the PCR product in concentration, and the permanent power electrophoresis of 12W separated in 1-1.5 hour.The plate that will have PAGE glue is put into concentration, and to be that 10% glacial acetic acid solution jog is moving all decolour to glue; With ultrapure water rinsing offset plate; Add the about 30min of colour developing liquid jog; Get offset plate express developed with ultrapure water; Transfer to offset plate in the refrigerative colour developing liquid and jog to being with line to occur; Offset plate moved quickly in 10% the glacial acetic acid solution; Clean sheet glass twice with ultrapure water; Offset plate is placed seasoning under the room temperature; End reaction promptly obtains the polymorphism collection of illustrative plates of turbot FF0922 locus.
The technology of the present invention has following characteristics:
(1) the present invention can obtain the heritable variation collection of illustrative plates of the FF0922 genetic marker locus of turbot efficiently, and method is easy, and the gained result can detect each individual genotype of turbot intuitively.
(2) the present invention be mainly used in genetic marker between turbot colony, genealogical identification, construction of genetic atlas and with chain character screening in this site etc.
(3) the FF0922 genetic marker locus that the present invention detected is the new turbot microsatellite marker that autonomous constructed dna enriched library is found.
(4) through after the condition optimizing, this primer can amplify the FF0922 microsatellite marker accurately, and the PCR product presents better polymorphism in turbot colony is detected.
(5) microsatellite marker meets the Mendelian inheritance pattern for other molecular genetic marker technique, is codominant inheritance, therefore, can discriminate individuals be homozygote or heterozygote state.
Description of drawings:
Fig. 1: the FF0922 primer of invention is to the detection collection of illustrative plates of 30 individualities of turbot:
Numbering 1-30 is 30 individualities of turbot, and M is a 50bp DNA Ladder standard molecular weight.
Embodiment:
Be described in detail the present invention in conjunction with the accompanying drawings at the little satellite core sequence of turbot FF0922 dna molecular genetic marker technological method below by embodiment.
At first extract turbot genomic dna and diluted for use; Utilize the microsatellite locus sequence that contains in the turbot genomic library that has made up again, in its sequence design specific primers at both ends; Use this primer that individual genomic dna in different geographical populations of turbot or the colony is carried out pcr amplification then, the PCR product is detected; The band that utilizes product to occur is analyzed, and determines the genotype that each is individual, and obtains the genetic polymorphism collection of illustrative plates in this site of turbot.
The extraction of turbot genomic dna: a, get the muscle tissue 50-100mg of fresh sample, in the 1.5mL centrifuge tube of packing into.B, every pipe add DNA extraction damping fluid [0.4mol/L, NaCl, 10mmol/L Tris-HCl (pH 8.0), 2mmol/L EDTA (pH 8.0), 1%SDS, 20 μ g/ml Pancreatic RNases] 400 μ L, will organize as far as possible to shred.Add 10%SDS 40 μ L in C, the every pipe, add 8 μ L Proteinase Ks (20mg/ml) again, mixing, 55～65 ℃, 3 hours (per therebetween half an hour mixing 1 time) digests to solution and clarifies.D, add 300ul 6mol/L NaCl, high-speed mixing is 30 seconds on vortex mixer, and centrifugal 30 minutes of 12000rpm moves supernatant 400ul in another centrifuge tube.E, adding equal-volume 400ul phenol: chloroform: primary isoamyl alcohol (volume ratio is 25: 24: 1), slowly put upside down mixing 8 minutes, the centrifugal 5-10 of 12000rpm minute, get supernatant 350ul and in clean centrifuge tube, (can repeat the operation of the 5th step according to circumstances).F, add equal-volume 350ul Virahol, mixing, put-20 ℃ 1 hour, 12000rpm is centrifugal 15 minutes then, abandons supernatant.The concentration that g, every pipe add-20 ℃ of precoolings is 70% ethanol 650ul, slowly puts upside down several times, carefully outwells ethanol (careful precipitation is lost), and repeated washing once removes ethanol then, dries up 5-10 minute.H, fill dissolving with aqua sterilisa after, quantitatively that its concentration that is diluted to 50ng/ul is standby.
2. the design of micro-satellite primers:
On the basis of the little satellite enrichment of turbot genomic library, obtain little satellite core sequence of FF0922 and two ends conserved sequence thereof and be:
1 ATGTGCTGTG?TCAGACTGTT?TATCTGTGAG?AAAACAGGCA?ATCAGAGAGG
51 GCGCTCACAT?ATTATTGAAA?TGACATGAAA?TCCATGTTAC?TCCACACTCA
101?AGTCGTATAT?GCAAGCTAGA?ATGGTGAATC?TCTTCTTGTC?TCCTACAGAC
151?AGACAAACAC?ACACACACAC?ACACACACAC?ACACACACAC?ACAAACACAT
201?ACACAAAACA?AGACCAACAG?TGACATGATA?TAACATGATC?ACCAAATCAC
251?AGAAACTGTA?GGGGAGAAGG?GGAAAAAAAG?CTAATAAAGC?TATAGCAAAA
301?TTAGAACTGT?GCTGTAGGCA?GATCCAGTGT?CTCCAGTAAT?CTACATAAGC
351?AGCAGTTGTC?GT；
Utilize SSR Hunter software to seek and analyze little satellite core sequence, according to the sequence of microsatellite DNA both sides at the high conservative of same species with respect to core sequence, in its design specific primers at both ends be: normal chain 5 '-TGTGAGAAAACAGGCAATC-3 ', minus strand 5 '-CAGTTTCTGTGATTTGGTGAT-3 '; Annealing temperature is 60 ℃ when using primer.
3.PCR amplification:
The application of sample amount is as follows: turbot genomic dna (50ng/ul), 2u l; 10 * PCR buffer, 2.5ul; Mg
2+(25mmol/ul), 2ul; Taq enzyme (5U/ul), 0.2ul; DNTP (each 10mmol/l) each 0.5; Present method is at the primer each (10Pmol/L) of the little satellite core sequence of FF0922 two ends design, 2ul; Add ddH2O to 25ul.The program that the PCR instrument is set when using this primer is 94 ℃ of sex change 4min; 94 ℃ of 30sec, 60 ℃ of 50sec, 72 ℃ of 50sec, 30 circulations; 72 ℃ are extended 7min, 4 ℃ of preservations.
After 4.PCR the detection of product: PCR reaction finishes, is 8% denaturing polyacrylamide gel with the PCR product in concentration, the permanent power electrophoresis of 12W separated in 1-1.5 hour. and the concentration that the plate that will have PAGE glue is put into the new preparation of 1L is 10% glacial acetic acid solution, shake about 30min gently, all decolour to glue; With 1L ultrapure water rinsing offset plate, totally 2 times, each 2min; Add the moving 30min of 1L colour developing liquid jog; Get the offset plate two sides express developed to remove the staining fluid of gel surface with ultrapure water; Transfer to offset plate in the 1L refrigerative colour developing liquid fast and shake gently, (about 5-10min) occur until the band line; Offset plate moved quickly in 10% the glacial acetic acid solution jog 3-5min; Cleaning sheet glass twice with ultrapure water. end reaction promptly obtains the polymorphism collection of illustrative plates of turbot FF0922 heredity as shown in Figure 1.
Sequence table
SEQUENCE?LISTING
＜110〉Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
＜120〉exercise question utilizes the detection method of Auele Specific Primer to turbot FF0922 microsatellite marker
<141>2009-11-18
<160>3
<170>PatentIn?version?3.1
<210>1
<211>19
<212>DNA
<213>Artificial?Sequence
<220>
<221>prim_bind
<222>(1)...(19)
＜223〉be used to the to increase forward primer sequence of FF0922
<400>1
<210>2
<211>21
<212>DNA
<213>Artificial?Sequence
<220>
<221>prim_bind
<222>(1)...(21)
＜223〉be used to the to increase reverse primer sequence of FF0922
<400>2
<210>3
<211>362
<212>DNA
＜213〉turbot (Scophthalmus maximus L.)
<400>3
1 ATGTGCTGTG?TCAGACTGTT?TATCTGTGAG?AAAACAGGCA?ATCAGAGAGG
51 GCGCTCACAT?ATTATTGAAA?TGACATGAAA?TCCATGTTAC?TCCACACTCA
101?AGTCGTATAT?GCAAGCTAGA?ATGGTGAATC?TCTTCTTGTC?TCCTACAGAC
151?AGACAAACAC?ACACACACAC?ACACACACAC?ACACACACAC?ACAAACACAT
201?ACACAAAACA?AGACCAACAG?TGACATGATA?TAACATGATC?ACCAAATCAC
251?AGAAACTGTA?GGGGAGAAGG?GGAAAAAAAG?CTAATAAAGC?TATAGCAAAA
301?TTAGAACTGT?GCTGTAGGCA?GATCCAGTGT?CTCCAGTAAT?CTACATAAGC
351?AGCAGTTGTC?GT
Claims (1)
1. a detection method of utilizing Auele Specific Primer to turbot FF0922 microsatellite marker is characterized in that its technological operation program is: at first extract turbot DNA; Utilize the microsatellite DNA core sequence of FF0922 clone in the turbot genomic library again, in its sequence design specific primers at both ends; Use this primer that the genomic dna of Different Individual in different geographical populations of turbot or the colony is carried out pcr amplification then, the PCR product is carried out denaturing polyacrylamide gel electrophoresis detect; The band that utilizes product to occur is analyzed, and determines the genotype that each is individual, and obtains the genetic diversity variation collection of illustrative plates in turbot FF0922 core sequence district;
The turbot genomic dna of described extraction dilutes and is 50ng/ul, and adding 2ul reaction cumulative volume in its each PCR reaction is 25ul;
Described pcr amplification: its application of sample parameter is: each PCR reaction cumulative volume is 25ul, comprises 100ng turbot genomic dna; 10 * PCR buffer, 2.5ul; Mg
2+, 2mmol/L; Taq enzyme 1U; Each 0.2mmol/L of dNTP; Each 0.2umol/L of primer of the little satellite core sequence of FF0922 two ends design; Add ddH
2O to 25ul; The program that the PCR instrument is set when using this primer is 94 ℃ of sex change 4min; 94 ℃ of 30sec, 60 ℃ of 50sec, 72 ℃ of 50sec, 30 circulations; 72 ℃ are extended 7min, 4 ℃ of preservations;
Described PCR product detects: is 8% denaturing polyacrylamide gel with the PCR product in concentration, and the permanent power electrophoresis of 12W separated in 1-1.5 hour; It is that 10% glacial acetic acid solution shakes to glue and all decolours that the plate that will have PAGE glue is put into concentration; Float Xian's offset plate 2 times with ultrapure water; Adding colour developing liquid shakes gently; Get the offset plate two sides express developed to remove the staining fluid of gel surface with ultrapure water; Offset plate is transferred in the refrigerative colour developing liquid fast until the appearance of band line; Offset plate is moved quickly into jog 3-5min in 10% glacial acetic acid solution; Clean sheet glass with ultrapure water; Offset plate is placed seasoning under the room temperature; Can obtain the genetic polymorphism collection of illustrative plates of turbot FF0922 locus;
It is characterized in that described: little satellite core sequence of FF0922 and two ends conserved sequence thereof are:
1ATGTGCTGTG?TCAGACTGTT?TATCTGTGAG?AAAACAGGCA?ATCAGAGAGG
51GCGCTCACAT?ATTATTGAAA?TGACATGAAA?TCCATGTTAC?TCCACACTCA
101AGTCGTATAT?GCAAGCTAGA?ATGGTGAATC?TCTTCTTGTC?TCCTACAGAC
151AGACAAACAC?ACACACACAC?ACACACACAC?ACACACACAC?ACAAACACAT
201ACACAAAACA?AGACCAACAG?TGACATGATA?TAACATGATC?ACCAAATCAC
251AGAAACTGTA?GGGGAGAAGG?GGAAAAAAAG?CTAATAAAGC?TATAGCAAAA
301TTAGAACTGT?GCTGTAGGCA?GATCCAGTGT?CTCCAGTAAT?CTACATAAGC
351AGCAGTTGTC?GT；
The specific primer sequence of its core sequence is respectively: normal chain 5 '-TGTGAGAAAACAGGCAATC-3 ', minus strand 5 '-CAGTTTCTGTGATTTGGTGAT-3 '; The annealing temperature of this primer is 60 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102309061A CN101705296B (en) | 2009-11-20 | 2009-11-20 | Detection method of turbot FF0922 microsatellite marker by utilizing specific primers |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102309061A CN101705296B (en) | 2009-11-20 | 2009-11-20 | Detection method of turbot FF0922 microsatellite marker by utilizing specific primers |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101705296A true CN101705296A (en) | 2010-05-12 |
| CN101705296B CN101705296B (en) | 2011-10-26 |
Family
ID=42375548
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102309061A Expired - Fee Related CN101705296B (en) | 2009-11-20 | 2009-11-20 | Detection method of turbot FF0922 microsatellite marker by utilizing specific primers |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101705296B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880719A (en) * | 2010-07-16 | 2010-11-10 | 中国水产科学研究院黄海水产研究所 | Microsatellite multi-PCR (Polymerase Chain Reaction) method for turbot paternity test |
| CN102586454A (en) * | 2012-03-14 | 2012-07-18 | 中国水产科学研究院黄海水产研究所 | Scophthalmus maximus T170G single nucleotide polymorphic marking detection method |
| CN103627807A (en) * | 2013-12-06 | 2014-03-12 | 广东省昆虫研究所 | Sacalia bealei microsatellite marked specific primer and detection method |
-
2009
- 2009-11-20 CN CN2009102309061A patent/CN101705296B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880719A (en) * | 2010-07-16 | 2010-11-10 | 中国水产科学研究院黄海水产研究所 | Microsatellite multi-PCR (Polymerase Chain Reaction) method for turbot paternity test |
| CN101880719B (en) * | 2010-07-16 | 2012-09-05 | 中国水产科学研究院黄海水产研究所 | Microsatellite multi-PCR (Polymerase Chain Reaction) method for turbot paternity test |
| CN102586454A (en) * | 2012-03-14 | 2012-07-18 | 中国水产科学研究院黄海水产研究所 | Scophthalmus maximus T170G single nucleotide polymorphic marking detection method |
| CN103627807A (en) * | 2013-12-06 | 2014-03-12 | 广东省昆虫研究所 | Sacalia bealei microsatellite marked specific primer and detection method |
| CN103627807B (en) * | 2013-12-06 | 2015-05-20 | 广东省昆虫研究所 | Sacalia bealei microsatellite marked specific primer and detection method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101705296B (en) | 2011-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101705295B (en) | Detection method of turbot FF0901 microsatellite marker by utilizing specific primers | |
| CN101294217B (en) | Blue crab ptssr17 microsatellite DNA marker testing technique | |
| CN101705296B (en) | Detection method of turbot FF0922 microsatellite marker by utilizing specific primers | |
| CN105624322A (en) | Method for developing SSR (Simple Sequence Repeat) molecular mark of nibea albiflora for population identification | |
| CN101294218B (en) | Blue crab ptssr36 microsatellite DNA marker testing technique | |
| CN107937395B (en) | Microsatellite molecular marker for polymorphism of high-sea swimming crabs, and identification method and application thereof | |
| CN104419705A (en) | SNP (single nucleotide polymorphism) marker and application thereof | |
| CN105177142B (en) | A kind of strain line hippocampus microsatellite marker and its screening technique | |
| KR102074139B1 (en) | Genetic markers and methods for identifying species belong to Cobitidae | |
| CN102586451A (en) | Method for matching and breeding jian carps | |
| CN100516235C (en) | Method for detecting micro-satellite marker LJ_liu of Lateolabrax japonicus | |
| Kalyuzhnaya et al. | Development and application of polymerase chain reaction primers based on fhcD for environmental detection of methanopterin‐linked C1‐metabolism in bacteria | |
| CN102277438B (en) | Method for detecting EcSSR1024 microsatellite DNA marker in exopalaemon carinicauda | |
| CN103484454A (en) | Sebastes schlegeli microsatellite DNA (Deoxyribonucleic Acid) molecular marker | |
| CN102851357A (en) | Detection method of microsatellite marker of clam | |
| Jones et al. | A quantitative SMRT cell sequencing method for ribosomal amplicons | |
| CN106755403A (en) | A kind of method that utilization specific primer group differentiates two class squilla oratoria populations | |
| CN106148541A (en) | SNP primer and screening technique for the screening of red fin east seed | |
| CN101967519A (en) | Method for identifying fx151 genetic variation map by using litopenaeus vannamei boone fx151 microsatellite DNA marker | |
| Lim et al. | Analysis and selection of microsatellites markers for individual traceability system in hanwoo | |
| CN112813171A (en) | MHC gene primer for cupreous rotundifolia fish and application thereof | |
| CN107058477B (en) | Method for detecting Sp6 microsatellite marker by using specific primer pair | |
| CN108913784A (en) | A kind of detection method of exopalaemon carinicauda EC12 SNP marker | |
| CN110616270A (en) | COI gene sequence-based molecular identification method of beta and beta | |
| CN104818341B (en) | A kind of saladifish microsatellite marker Epinmoss detection primer and method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20111026 Termination date: 20121120 |