CN105177158A - Microsatellite triple ternary PCR detection method of marsupenaeus japonicas - Google Patents
Microsatellite triple ternary PCR detection method of marsupenaeus japonicas Download PDFInfo
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- CN105177158A CN105177158A CN201510664366.3A CN201510664366A CN105177158A CN 105177158 A CN105177158 A CN 105177158A CN 201510664366 A CN201510664366 A CN 201510664366A CN 105177158 A CN105177158 A CN 105177158A
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Abstract
The invention discloses a microsatellite triple ternary PCR detection method of marsupenaeus japonicas. The method includes the steps that three pairs of PCR primers are designed and synthesized; the total DNA is extracted; combination, a system and a reaction program of the ternary PCR reactions are provided; PCR products are detected. The efficiency of the microsatellite triple ternary PCR detection method of marsupenaeus japonicas can be tripled compared with an original method. The method has the advantages that the cost and time are saved, and the method is efficient, economical, high in repeatability, simple, feasible, beneficial to detection and the like and can be applied and popularized with respect to breeding management, family identification, individual recognition, genetic relationship identification, improved breed breeding, linkage map construction, genetic diversity detection and the like of marsupenaeus japonicas.
Description
Technical field
The invention belongs to the molecule marker ancillary technique of molecular biology genetic breeding, be specifically related to a kind of Marsupenaeus japonicus micro-satellite triple PCR detection method.
Background technology
Marsupenaeus japonicus (Penaeusjaponicus) is distributed widely in the Indian Ocean, West Pacific region, and all there is distribution in the marine site such as the coastal and China's southeastern coast of Japan, is the important cultivation object in China's Coastal Areas.Utilizing molecule marker to assist is one of important channel of Marsupenaeus japonicus fine-variety breeding, and current micro-satellite is a kind of molecule marker of widespread use.Micro-satellite is distributed widely in genome, and polymorphism is high, good stability, codominant inheritance.Be widely used in the numerous areas such as pedigree qualification, individual recognition, gene linkage analysis, genetic linkage maps structure, Diversity Detection.Conventional micro-satellite inspection technique is single primer Standard PCR, and efficiency is low, and multiplex PCR increases multiple site in same reaction simultaneously, greatly improves efficiency, reduces sample waste, accelerate detection procedure, greatly saved the advantages such as experimental cost.Micro-satellite multiple PCR technique is widely used in aquatic animal, is mainly used in paternity test and Diversity Detection.The relevant report of multiplex PCR detection technique is there is not yet in Marsupenaeus japonicus.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of triple PCR detection method of Marsupenaeus japonicus microsatellite marker, this invention constructs triple PCR detection system and a trace routine on the basis of a large amount of microsatellite molecular marker, achieve detect 3 microsatellite locus simultaneously in a PCR reaction, improve 3 times with conventional PCR method phase specific efficiency, acquired results can reflect the genetic polymorphism of the multiple microsatellite locus of Marsupenaeus japonicus simultaneously, for the breeding of Marsupenaeus japonicus molecular marker assisted selection provides efficient and micro-satellite inspection technique accurately.
For realizing object of the present invention, the present invention adopts following technical proposals to be achieved: a kind of Marsupenaeus japonicus micro-satellite triple PCR detection method, and it comprises: (1) design, the heavy PCR primer of synthesis 3; (2) STb gene is extracted; (3) triple PCR reaction; (4) electrophoresis detection PCR primer; Described 3 heavy PCR reactions comprise triple PCR reaction system and triple PCR response procedures, and the primer of described triple PCR reaction system is the combination of SEQ036, SEQ031 and SEQ043, and the sequence of described triple PCR primer is as follows:
SEQ036 forward primer sequence: 5 '-AAGGGAATTTGAGTAGAGTCTG-3'; SEQ036 reverse primer sequences: 5'-GTTACATGCGAGTTGCTATT-3'
SEQ031 forward primer sequence: 5'-ACGCTGGTTTCATTGGGATT-3'; SEQ031 reverse primer sequences: 5'-AAATGTGGGAGGGCGAAA-3'
SEQ043 forward primer sequence: 5'-ATTGCTGTCGGGATGAGA-3'; SEQ043 reverse primer sequences: 5'-TGGTTGTTCGGAAGAGGT-3'
Described triple PCR reaction system is: 10 × Buffer2 μ L; 25mMMg
2+2 μ L; 10mMdNTP2 μ L, 10 μMs of each 1 μ L of 3 couples of amplimers SEQ036, SEQ031 and SEQ043; 5U/ μ LTaqDNA polysaccharase 0.1 μ L; 100ng/ μ L Marsupenaeus japonicus STb gene: 1 μ L; Sterilizing distilled water complements to 25 μ L.
Described triple PCR response procedures is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 40s, 51 DEG C of annealing 1min, 72 DEG C extend 1min, carry out 28 circulations altogether; Last 72 DEG C extend 5min; Preserve for 4 DEG C and terminate reaction.
Step of the present invention (4) adopts 8% denaturing polyacrylamide gel electrophoresis to detect PCR primer.
Compared with prior art, beneficial effect of the present invention:
1, the present invention carries out the Marsupenaeus japonicus microsatellite PCR amplification of 3 times respectively by needing and detects for 3 times, is integrated into 3 heavy PCR reaction systems once and a product detection, substantially increases the detection efficiency of Marsupenaeus japonicus microsatellite marker.Utilize this 3 heavy PCR reaction system to carry out the micro-satellite of Marsupenaeus japonicus to detect, greatly can save PCR and electrophoresis reagents, experiment consumptive material, reduce electrophoresis detection time and cost.
2,3 heavy PCR of the present invention react gained micro-satellite product fragment significant difference, can avoid different loci product clip size close to time the shortcoming that cannot distinguish.
3, the present invention can detect in each period of growing Marsupenaeus japonicus, is conducive to Marsupenaeus japonicus family and the colony of selecting to possess good character in Marsupenaeus japonicus breeding process.
4, the present invention can apply in the analysis of Marsupenaeus japonicus population genetic diversity, parentage identification, paternity test, breeding management, fine-variety breeding.
Accompanying drawing explanation
Fig. 1 is Marsupenaeus japonicus 3 heavy pcr amplification product polyacrylamide gel electrophoresis figure.
In figure, M is markerD2000, and 1 is the amplification of described 3 pairs of primers, and 2,3,4 is the amplification of described 3 pairs of primer combination of two, and 5,6,7 is the amplification of described list to primer.
Embodiment
Below in conjunction with drawings and Examples, specifically describe content of the present invention.Should be appreciated that enforcement of the present invention is not limited to the following examples, any pro forma accommodation make the present invention or change all will fall into scope.
Embodiment 1
(1), design of primers and combination
Utilize TandemRepeatFinder to search microsatellite sequence from Random clones sequencing sequence, the flanking sequence PremierPrimer choosing core repeat sequence both sides complete designs primer.MPprimer carries out the combination evaluation between primer, and the absolute value getting △ G is less than 3, reduces the probability that secondary structure is formed, and improves PCR atopic and reaction efficiency.By to the combination between multipair micro-satellite primers, Marsupenaeus japonicus multiplex PCR system combinations, PCR reaction conditions and response procedures are optimized, finally set up the heavy PCR reaction system of the micro-satellite 3 of a kind of Marsupenaeus japonicus of the present invention, response procedures and detection method.3 pairs of Marsupenaeus japonicus primers selected by the present invention are:
SEQ036 forward primer sequence: 5 '-AAGGGAATTTGAGTAGAGTCTG-3'
SEQ036 reverse primer sequences: 5'-GTTACATGCGAGTTGCTATT-3'
SEQ031 forward primer sequence: 5'-ACGCTGGTTTCATTGGGATT-3'
SEQ031 reverse primer sequences: 5'-AAATGTGGGAGGGCGAAA-3'
SEQ043 forward primer sequence: 5'-ATTGCTGTCGGGATGAGA-3'
SEQ043 reverse primer sequences: 5'-TGGTTGTTCGGAAGAGGT-3'
(2), Marsupenaeus japonicus total DNA extraction
Get muscle tissue and be about 100mg, put into 1.5ml centrifuge tube, add TE (10mMTris-Cl, 100mMEDTA) the 475 μ l of pH8.0, scissors shreds, and adds 10%SDS solution 20 μ l, adds 20mg/ml Proteinase K 5 μ l, mixing, 55 DEG C of digestion 2.5-3 hour, to inorganization block.Saturated phenol, phenol is balanced: chloroform: primary isoamyl alcohol=25:24:1 (volume ratio) mixing solutions, each extracting of chloroform are once with Tris-Cl, the centrifugal 10min of each mixing 10min, 12000rpm, gets supernatant, 1/25 volume 5MNaCl is reset and added finally, 2.5 times of volumes-20 DEG C of freezing dehydrated alcohol precipitation DNA, leave standstill 10min, centrifugal 10min, precipitation washes 2 times with 70% ethanol, drying at room temperature, dissolves with sterilizing distilled water 100 μ l, and 1% sepharose detects DNA quality.
(3), the heavy PCR reaction system of Marsupenaeus japonicus 3
The heavy PCR reaction system of Marsupenaeus japonicus 3 is: 10 × Buffer2 μ L; 25mMMg
2+2 μ L; 10mMdNTP2 μ L, 10 μMs of each 1 μ L of 3 couples of amplimers SEQ036, SEQ031 and SEQ043; 5U/ μ LTaqDNA polysaccharase 0.1 μ L; 100ng/ μ L Marsupenaeus japonicus STb gene: 1 μ L; Sterilizing distilled water complements to 25 μ L.
(4), the heavy PCR response procedures of Marsupenaeus japonicus 3
The heavy PCR response procedures of Marsupenaeus japonicus 3 is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 40s, 51 DEG C of annealing 1min, 72 DEG C extend 1min, carry out 28 circulations altogether; Last 72 DEG C extend 5min; Preserve for 4 DEG C and terminate reaction.
(5), the detection of pcr amplification product
PCR reacts end, after product is mixed with loading buffer 1:1, through 8% denaturing polyacrylamide gel electrophoresis, and electrophoresis apparatus power 12W, electrophoresis 1-2.5h.By offset plate silver dye colour developing after electrophoresis, record acquired results.
Fig. 1 is Marsupenaeus japonicus 3,2,1 heavy pcr amplification denaturing polyacrylamide gel electrophoresis figure.In figure, M is marker, and 1 is the amplification of described 3 pairs of primers, and 2,3,4 is the amplification of combination of two, and 5,6,7 is the amplification of single primer.Experimental result shows that 3 pairs of combination of primers amplifications are clear, and resolving power is high.
According to the combination of primers of 3 heavy PCR of Marsupenaeus japonicus microsatellite marker provided by the present invention and the single individuality of detection method rapid detection Marsupenaeus japonicus in the heritable variation of 3 microsatellite locus and polymorphism, can be applicable to the aspects such as the parentage identification of Marsupenaeus japonicus, paternity test, genetic marker screening, genetic linkage maps structure further.
Above example only illustrates technical scheme of the present invention, be to be understood that, for a person skilled in the art, amendment and/or accommodation are made to above-described embodiment or adopts equivalent replacement scheme to be obvious, all can not depart from the essence of spirit of the present invention, the technical term occurred in the present invention, for elaboration of the present invention and understanding, can not make restriction to the present invention.
Claims (2)
1. a Marsupenaeus japonicus micro-satellite triple PCR detection method, is characterized in that, comprises the steps: (1) design, the heavy PCR primer of synthesis 3; (2) STb gene is extracted; (3) triple PCR reaction; (4) electrophoresis detection PCR primer; Described 3 heavy PCR reactions comprise triple PCR reaction system and triple PCR response procedures,
The primer of described step (3) triple PCR reaction system is the combination of SEQ036, SEQ031 and SEQ043, and the sequence of described triple PCR primer is as follows:
SEQ036 forward primer sequence: 5 '-AAGGGAATTTGAGTAGAGTCTG-3'; SEQ036 reverse primer sequences: 5'-GTTACATGCGAGTTGCTATT-3'
SEQ031 forward primer sequence: 5'-ACGCTGGTTTCATTGGGATT-3'; SEQ031 reverse primer sequences: 5'-AAATGTGGGAGGGCGAAA-3'
SEQ043 forward primer sequence: 5'-ATTGCTGTCGGGATGAGA-3'; SEQ043 reverse primer sequences: 5'-TGGTTGTTCGGAAGAGGT-3'
Described triple PCR reaction system is: 10 × Buffer2 μ L; 25mMMg
2+2 μ L; 10mMdNTP2 μ L, 10 μMs of each 1 μ L of 3 couples of amplimers SEQ036, SEQ031 and SEQ043; 5U/ μ LTaqDNA polysaccharase 0.1 μ L; 100ng/ μ L Marsupenaeus japonicus STb gene: 1 μ L; Sterilizing distilled water complements to 25 μ L;
Described triple PCR response procedures is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 40s, 51 DEG C of annealing 1min, 72 DEG C extend 1min, carry out 28 circulations altogether; Last 72 DEG C extend 5min; Preserve for 4 DEG C and terminate reaction.
2. Marsupenaeus japonicus micro-satellite triple PCR detection method according to claim 1, is characterized in that, described step (4) adopts 8% denaturing polyacrylamide gel electrophoresis to detect PCR primer.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107190098A (en) * | 2017-07-27 | 2017-09-22 | 四川省自然资源科学研究院 | The triple PCR system detection method of a set of giant panda microsatellite locus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1680601A (en) * | 2005-01-25 | 2005-10-12 | 孔杰 | Micro-satellite triple PCR family identification of China peneid |
| CN102399776A (en) * | 2011-09-02 | 2012-04-04 | 中国水产科学研究院黄海水产研究所 | Microsatellite marker quadruple PCR primer for identifying Penaeus chinesis family and identification method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1680601A (en) * | 2005-01-25 | 2005-10-12 | 孔杰 | Micro-satellite triple PCR family identification of China peneid |
| CN102399776A (en) * | 2011-09-02 | 2012-04-04 | 中国水产科学研究院黄海水产研究所 | Microsatellite marker quadruple PCR primer for identifying Penaeus chinesis family and identification method |
Non-Patent Citations (4)
| Title |
|---|
| LI Y ET AL.: "Development of two microsatellite multiplex systems for black tiger shrimp Penaeus monodon and its application in genetic diversity study for two populations", 《AQUACULTURE》 * |
| 孔沛球等: "不同地理群体日本囊对虾遗传多样性微卫星DNA分析", 《水产科学》 * |
| 栾生等: "日本囊对虾(Marsupenaeus japonicus)基因组微卫星特征分析", 《自然科学进展》 * |
| 郭慧等: "三个野生群体日本囊对虾遗传多样性的SSR分析", 《生物技术通报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107190098A (en) * | 2017-07-27 | 2017-09-22 | 四川省自然资源科学研究院 | The triple PCR system detection method of a set of giant panda microsatellite locus |
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