CN1233845C - Chinese prawn B683 microsatellite label detecting technique - Google Patents
Chinese prawn B683 microsatellite label detecting technique Download PDFInfo
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- CN1233845C CN1233845C CN 02135713 CN02135713A CN1233845C CN 1233845 C CN1233845 C CN 1233845C CN 02135713 CN02135713 CN 02135713 CN 02135713 A CN02135713 A CN 02135713A CN 1233845 C CN1233845 C CN 1233845C
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Abstract
The present invention relates to a detection technology of a Chinese prawn B683 microsatellite mark, which comprises: firstly, extracting Chinese prawn genomicDNA; secondly, designing specific primers at both ends of a microsatellite sequence contained in the Chinese prawn genomic library, carrying out PCR amplification to the Chinese prawn genomic DNA, and electrophoretic separation is carried out to the obtained PCR product in denatured polyacrylamide gel; thirdly, analyzing the strip appearing on the product to obtain a genetic polymorphism map of the Chinese prawn. The present invention is applicable to the detection technologies of a group genetic mark, genealogical identification, genetic map establishment, etc. of the Chinese prawn, and can detect the genotype of the individual Chinese prawn conveniently, quickly, accurately and visually.
Description
Affiliated technical field
The invention belongs to Chinese prawn dna molecular genetic marker technology, is a kind of technological method that detects Chinese prawn in B683 site genetic polymorphism.
Background technology
Before the present invention makes, similar research report is arranged both at home and abroad, (2001a such as the Xu Peng of ocean institute of the domestic Chinese Academy of Sciences, " recombinant clone that contains little satellite with PCR method rapid screening Chinese prawn " and the papers such as " screenings of Chinese prawn microsatellite DNA " 2001b) delivered, once reported the structure in Chinese prawn portion gene group library, the contents such as screening that contain microsatellite sequence, and little satellite core sequence is estimated according to the standard that Weber (1990) proposes, but his these results of study only limit to study the acquisition of microsatellite sequence, do not have further application and development.Institut Francais de Recherche Pour L'Exploitation de la Mer-ifremer (IFREMER) found 10 microsatellite markers in 1999 from blue prawn population, from tigar prawn, find 3 microsatellite markers, brought into play effect when these are marked at the different family that identification raises together with, but do not seen and announce microsatellite marker sequence and primer sequence.At present, do not see the report of aspects such as structure, specificity genetic marker and the genealogical identification application of Chinese prawn microsatellite polymorphism collection of illustrative plates as yet.
Summary of the invention
The present invention its objective is the dna molecular genetic marker technology that proposes a kind of Chinese prawn B683 sequence, the main utilization contained the little satellite core sequence of B683 in the Chinese prawn portion gene group library of setting up, carrying out PCR in its design specific primers at both ends detects, thereby detect of the heritable variation of each individuality of Chinese prawn apace in this little satellite district, obtain this primer to the heritable variation collection of illustrative plates of Chinese prawn, detect each individual genotype intuitively by collection of illustrative plates in B683 core sequence district.
The present invention finishes according to following operational aspect: at first extract Chinese prawn genomic dna and diluted for use; Utilize the little satellite core sequence that contains the B683 clone in the Chinese prawn genomic library again, in its sequence design specific primers at both ends; Use this primer that individual genomic dna in different proles of Chinese prawn or the Chinese prawn group is carried out pcr amplification then, the PCR product is carried out denaturing polyacrylamide gel detect; The band that utilizes product to occur is analyzed, and determines the genotype that each is individual, thereby and obtains the polymorphism collection of illustrative plates of Chinese prawn in the heritable variation of B683 core sequence district height.
Extract the Chinese prawn genomic dna, its dilution is 50ng/ μ l, add 2 μ l in each PCR reaction, the reaction cumulative volume is 25 μ l.
The specific primer sequence of the little satellite core sequence of B-683 is respectively: normal chain 5 '-ACA CTCACT TAT GTC ACA CTG C-3 ', and minus strand 5 '-TACA CAC CAA CAC TCA ATC TCC-3 ', the annealing temperature when using this primer is 64 ℃.
The application of sample parameter of pcr amplification is: each PCR reaction cumulative volume is 25 μ l, comprises 100ng Chinese prawn genomic dna; 10X PCR Buffer, 2.5 μ l; Mg
++2.0mmol/L; Tag enzyme 1u; Each 0.1mmol/L of dNTP; Each 0.2 μ mol/L of primer of the present invention; Add ddH
2O to 25 μ l.The program parameter that the PCR instrument is set when using this primer is: 94 ℃ of sex change 2min; 94 ℃ of 40sec, 64 ℃ of 1min, 72 ℃, 1min, 25 circulations; 72 ℃ are extended 5min, 4 ℃ of preservations.
The detection of PCR product: at 8% denaturing polyacrylamide gel, the permanent power electrophoresis of 12W separated in 1-1.5 hour with the PCR product.With the sodium carbonate solution colour developing of offset plate by silver nitrate solution 25min → 3% of glacial acetic acid solution 20min → distilled water 6min → 0.1% of 10% several minutes, termination reaction can obtain the polymorphism collection of illustrative plates of Chinese prawn in the heritable variation of B683 locus height.
Use this primer and described technological method, can detect the heritable variation of each individuality of Chinese prawn, can read its genotype by the polyacrylamide gel electrophoresis collection of illustrative plates at the little satellite core area of B683, convenient, fast, accurate.Secondly, microsatellite marker meets the Mendelian inheritance pattern for other molecular genetic marker technique, is codominant inheritance, therefore, can discriminate individuals be homozygote and heterozygote state.
The present invention is applicable to application such as Chinese prawn population genetic mark, genealogical identification, genetic map construction.The PCR primer of B683 is a core of the present invention, presents the polymorphism of height heritable variation in the total group of Chinese prawn detects.
The technology of the present invention has following characteristics:
(1) the present invention can obtain the polymorphism collection of illustrative plates of heritable variation of the B683 genetic marker locus of Chinese prawn fast, and method is simple and efficient, and the gained result can detect the genotype of each individuality of Chinese prawn in this site intuitively;
(2) the present invention is mainly used in genetic marker, genealogical identification and construction of genetic atlas etc. between Chinese prawn colony.
Description of drawings:
Fig. 1 B683 primer of the present invention is to the detection collection of illustrative plates of 60 individualities of Chinese prawn, and numbering 1-60 is 60 individualities of Chinese prawn, and M is the DL2000 standard molecular weight.
Embodiment
Be described in detail the present invention at the little satellite core sequence of Chinese prawn B683 dna molecular genetic marker technological method below by embodiment.At first extract Chinese prawn genomic dna and diluted for use; Utilize again and contain the little satellite core sequence of B683 in the Chinese prawn genomic library, in its sequence design specific primers at both ends; Use this primer that individual genomic dna in different proles of Chinese prawn or the Chinese prawn group is carried out pcr amplification then, the PCR product is carried out polyacrylamide gel electrophoresis detect; The band that utilizes product to occur is analyzed, and determines the genotype that each is individual, thereby obtains the polymorphism collection of illustrative plates of Chinese prawn in the heritable variation in B683 core sequence district.
1, the extraction of Chinese prawn genomic dna: get prawn muscle tissue 100mg, put into the 1.5ml centrifuge tube after shredding, (10mmol/L Tris-CI, 10mmol/LEDTA) 500 μ l grind with grinding rod the TE solution of adding pH8.0.Add 10%SDS solution 25 μ l, mixing.Add 20mg/ml Proteinase K 4 μ l, mixing, 55 ℃ of digestion 2.5～3h.Re-distilled phenol extracting twice, each 10min, the centrifugal 5min of 12000 commentaries on classics/min gets supernatant; Phenol: chloroform (1: 1) extracting once, 10min, the centrifugal 5min of 12000 commentaries on classics/min gets supernatant; 5min of chloroform extracting, the centrifugal 5min of 5000g gets supernatant.The 5mol/L NaCl solution that adds 1/25 volume adds-20 ℃ dehydrated alcohol deposit D NA 15min of two volumes again behind the mixing; The DNA that chooses, the washing with alcohol with 70% tens of minutes makes the DNA drying, and is after fully dissolving with aqua sterilisa 500 μ l, quantitatively that its concentration that is diluted to 50ng/ μ l is standby.
2, the design of micro-satellite primers: on Chinese prawn genomic library basis, the sequence of utilizing the microsatellite DNA both sides is at the high conservative of same species with respect to core sequence, in the design specific primers at both ends of B683 core sequence, amplify the microsatellite DNA fragment in this site with it.Because little satellite core sequence mutation rate is higher relatively, causes the increase or the minimizing of microsatellite DNA core sequence multiplicity, i.e. the variation of microsatellite DNA sequence length, this is the main foundation that detects microsatellite polymorphism.The specific primer sequence at core sequence two ends, the little satellite of B683 of the present invention district is: normal chain 5 '-ACA CTC ACT TAT GTC ACA CTG C-3 ', minus strand 5 '-TACA CAC CAA CACTCA ATC TCC-3 '.When using this primer, its annealing temperature is 64 ℃.
3, pcr amplification:
Application of sample at first, the application of sample amount is as follows: Chinese prawn genomic dna (50ng/ μ l), 2 μ l; 10X PCR Buffer, 2.5 μ l; Mg
++(25mmol/L), 2 μ l; Tag enzyme (5u/ μ l), 0.2 μ l; DNTP (each 2.5mmol/L), 2 μ l; Primer of the present invention (each 10Pmol/ μ l), 2 μ l; Add aqua sterilisa to 25 μ l.Secondly, carry out the PCR reaction, its pcr amplification instrument program parameter is: 94 ℃ of sex change 2min; 94 ℃ of 40sec, 64 ℃ of 1min, 72 ℃, 1min, 25 circulations; 72 ℃ are extended 5min, 4 ℃ of preservations.
5, after the detection of PCR product: PCR reaction finishes, product is carried out electrophoresis in the gel of 8% sex change polyacrylamide, electrophoresis apparatus power be 12 watts, the electrophoretic time can stop about 1-1.5 hour.With the sodium carbonate solution colour developing of offset plate by silver nitrate solution 25min → 3% of glacial acetic acid solution 20min → distilled water 6min → 0.1% of 10% several minutes, termination reaction promptly obtained polymorphism collection of illustrative plates as shown in Figure 1.
Claims (5)
1. the detection technique of a Chinese prawn B683 microsatellite marker is characterised in that its technological operation program is: at first extract Chinese prawn genomic dna and diluted for use; Utilize the microsatellite DNA core sequence of B683 clone in the Chinese prawn genomic library again, in its sequence design specific primers at both ends; Use this primer that individual genomic dna in different proles of Chinese prawn or the Chinese prawn group is carried out pcr amplification then, the PCR product is carried out the sex change acrylamide gel detect; The band that utilizes product to occur is analyzed, and determines the genotype that each is individual, and obtains the genetic polymorphism variation collection of illustrative plates of Chinese prawn in B683 core sequence district.
2. the detection technique of a kind of Chinese prawn B683 microsatellite marker according to claim 1 is characterized in that extracting the Chinese prawn genomic dna, and its dilution is 50ng/ μ l, adds 2 μ l in each PCR reaction, and the reaction cumulative volume is 25 μ l.
3. the detection technique of a kind of Chinese prawn B683 microsatellite marker according to claim 1, the specific primer sequence that it is characterized in that the little satellite core sequence of B683 is respectively: normal chain 5 '-ACACTC ACT TAT GTC ACA CTG C-3 ', minus strand 5 '-TACA CAC CAA CAC TCA ATC TCC-3 ', the annealing temperature when using this primer is 64 ℃.
4. the detection technique of a kind of Chinese prawn B683 microsatellite marker according to claim 1 is characterized in that the application of sample parameter of pcr amplification is: each PCR reaction cumulative volume is 25 μ l, comprises 100ng Chinese prawn genomic dna; 10X PCR Buffer, 2.5 μ l; Mg
++2.0mmol/L; Tag enzyme 1u; Each 0.1mmol/L of dNTP; Present technique is at each 0.2 μ mol/L of primer of the little satellite core sequence of B683 two ends design; Add ddH
2O to 25 μ l.The program parameter that the PCR instrument is set when using this primer is: 94 ℃ of sex change 2min; 94 ℃ of 40sec, 64 ℃ of 1min, 72 ℃, 1min, 25 circulations; 72 ℃ are extended 5min, 4 ℃ of preservations.
5. the detection technique of a kind of Chinese prawn B683 microsatellite marker according to claim 1 is characterized in that the PCR product is detected: at 8% denaturing polyacrylamide gel, the permanent power electrophoresis of 12W separated in 1-1.5 hour with the PCR product.With the sodium carbonate solution colour developing of offset plate by silver nitrate solution 25min → 3% of glacial acetic acid solution 20min → distilled water 6min → 0.1% of 10% several minutes, termination reaction can obtain the genetic polymorphism collection of illustrative plates of Chinese prawn at the B683 locus.
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| CN 02135713 CN1233845C (en) | 2002-10-09 | 2002-10-09 | Chinese prawn B683 microsatellite label detecting technique |
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| CN 02135713 CN1233845C (en) | 2002-10-09 | 2002-10-09 | Chinese prawn B683 microsatellite label detecting technique |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101880721B (en) * | 2010-07-20 | 2012-09-26 | 中国水产科学研究院黄海水产研究所 | Method for detecting ES15 microsatellite markers of Chinese shrimps |
| CN101967519A (en) * | 2010-08-20 | 2011-02-09 | 中山大学 | Method for identifying fx151 genetic variation map by using litopenaeus vannamei boone fx151 microsatellite DNA marker |
| CN101967520B (en) * | 2010-08-20 | 2013-05-01 | 中山大学 | Method for identifying fx5 genetic variation diagram of Litopenaeus vannamei by fx5 microsatellite DNA marker |
| CN101942437B (en) * | 2010-08-23 | 2012-09-05 | 中山大学 | Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof |
| CN102304576B (en) * | 2011-08-24 | 2013-05-01 | 中国水产科学研究院黄海水产研究所 | Method for detecting EcSSR0003 microsatellite deoxyribonucleic acid (DNA) markers of palaemon carincauda holthuis |
| CN103416333B (en) * | 2013-07-31 | 2015-11-11 | 中国水产科学研究院黄海水产研究所 | A kind of construction method of the Environment of Litopenaeus vannamei Low basic population based on hereditary information and merit |
| CN105969873B (en) * | 2016-06-14 | 2020-07-10 | 中国科学院南海海洋研究所 | Litopenaeus vannamei osmotic pressure regulation related functional gene EST-SSR marker, and specific primer and detection method thereof |
| CN106755403A (en) * | 2016-12-22 | 2017-05-31 | 浙江海洋大学 | A kind of method that utilization specific primer group differentiates two class squilla oratoria populations |
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