CN100510103C - Micro-satellite triple PCR family identification of China peneid - Google Patents
Micro-satellite triple PCR family identification of China peneid Download PDFInfo
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- CN100510103C CN100510103C CNB2005100424029A CN200510042402A CN100510103C CN 100510103 C CN100510103 C CN 100510103C CN B2005100424029 A CNB2005100424029 A CN B2005100424029A CN 200510042402 A CN200510042402 A CN 200510042402A CN 100510103 C CN100510103 C CN 100510103C
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Abstract
The invention belongs to molecular genetic breeding technology for agricultural part. Augment parameter for three micro-satelloid primers in one PCR reaction system is obtained by adjusting PCR reaction system and using Touch-down PCR reaction process. Then ternary PCR technology is found.
Description
Technical field:
The invention belongs to the molecular biology part in the biology, concrete is the molecule ancillary technique of a genetic breeding, is at the cultivation of Crustin family and the family molecular recognition technology of developing;
Background technology:
The present invention has mainly utilized three micro-satellite primers RS1101, RS0683 and H081, relates to the development technique of these three micro-satellite primers, is that we develop at the design of the tumor-necrosis factor glycoproteins in the Crustin genome random sequencing sequence.
Summary of the invention:
The sequence of three micro-satellite primers is as follows:
RS1101 forward sequence is CGAGTGGCAGCGAGTCCT, and reverse sequence is TATTCCCACGCTCTTGTC
RS0683 forward sequence is ACACTCACTTATGTCACACTGC, and reverse sequence is TACACACCAACACTCAATCTCC
H081 forward sequence is ACAAACACATTCTGTCCATT, and reverse sequence is GATAGAGAGGTCAACAAACG
Make up this three primers, by adjusting the PCR reaction system, obtained the parameter that these three primers can increase simultaneously in same PCR reaction system, these parameters are: 10*Buffer 2.5 μ l (include 20mM Tris-HCl, 100mM KCl, 0.1mMEDTA, 1mM DTT, 0.5% Tween20), MgC l
22.5 μ l (2.5mM), dNTP 2.0 μ l (2.5mM), template DNA 2 μ l (50ng/ μ l), ddH
2O 12.1 μ l, Taq archaeal dna polymerase 0.4 μ l (5U/ μ l), primer RS 1101 forward and reverse each 1.0 μ l, primer RS 0683 forward and reverse each 1.5 μ l, forward and reverse each the 2.0 μ l of primer H081.The program that reaction is adopted is a Touch-down PCR program: carry out following procedure behind 94 ℃ of sex change 5min, and circulation 1:94 ℃ of sex change 40S, 66 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 6 circulations; The 2:94 ℃ of sex change 40S that circulate subsequently, 66 ℃ of annealing 1min, each circulation reduces by 0.5 ℃, and 72 ℃ are extended 1min, carry out 12 circulations; The 3:94 ℃ of sex change 40S that circulate again, 60 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 14 circulations.Last 72 ℃ are extended 10min..
Technical indicator: the RS1101 gene locus has 6 allelotrope, the RS0683 gene locus has 8 allelotrope, the H081 gene locus has 11 allelotrope, the parentage exclusion probability of Zu He this triple PCR technology (the accumulation parentage exclusion probabilities of three primers) is 0.9679 thus, and individual recognition power is 0.999327
Description of drawings
Fig. 1 is the amplification figure of RS1101 primer in 30 individualities, two allelotrope of the arrow among the figure has been indicated respectively the longest (No. 4 individualities) and the shortest (No. 27 individualities).
Fig. 2 is the amplification figure of RS0683 primer in 30 individualities, two allelotrope of the arrow among the figure has been indicated respectively the longest (No. 8 individualities) and the shortest (No. 23 individualities).
Fig. 3 is the amplification figure of H081 primer in 30 individualities, two allelotrope of the arrow among the figure has been indicated respectively the longest (No. 5 individualities) and the shortest (No. 1 individuality).
Fig. 4 is the triple PCR technology that combined by RS1101, RS0683 and three micro-satellite primers of the H081 amplification to male (♂) in 35191 familys and female (♀) parent and 10 filial generation individualities thereof, arrow has been indicated RS1101, RS0683 and the amplification of three primers of H081 in two parents from top to bottom respectively among the figure, 1-10 is two parents' doubtful filial generation, and wherein No. 10 individualities are identified the offspring who is not these two parents by this triple PCR technology.
Range of application:
Can pass through this triple PCR technology, three of the disposable acquisitions of pcr amplification of process usual manner The hereditary information of the Crustin individuality on the microsatellite locus, and then by aobvious between these Different Individual The difference of this hereditary information of showing is come they are distinguished and identify. If be used in the parentage identification, After the hereditary information of three microsatellite locus that obtained parent (Parent), we just can be right The offspring who raises together with carries out genetic analysis, and whether determine or get rid of these offsprings is sons of the parent that detects In generation, is individual.
Claims (1)
1, be used for the micro-satellite primers of Crustin family identification, it is characterized in that the sequence of three micro-satellite primers is:
RS1101 forward primer sequence is 5 '-CGAGTGGCAGCGAGTCCT-3 ', and the reverse primer sequence is 5 '-TATTCCCACGCTCTTGTC-3 ';
RS0683 forward primer sequence is 5 '-ACACTCACTTATGTCACACTGC-3 ', and the reverse primer sequence is 5 '-TACACACCAACACTCAATCTCC-3 ';
H081 forward primer sequence is 5 '-ACAAACACATTCTGTCCATT-3 ', and the reverse primer sequence is 5 '-GATAGAGAGGTCAACAAACG-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100424029A CN100510103C (en) | 2005-01-25 | 2005-01-25 | Micro-satellite triple PCR family identification of China peneid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100424029A CN100510103C (en) | 2005-01-25 | 2005-01-25 | Micro-satellite triple PCR family identification of China peneid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1680601A CN1680601A (en) | 2005-10-12 |
| CN100510103C true CN100510103C (en) | 2009-07-08 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100424029A Expired - Fee Related CN100510103C (en) | 2005-01-25 | 2005-01-25 | Micro-satellite triple PCR family identification of China peneid |
Country Status (1)
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| CN (1) | CN100510103C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101798570A (en) * | 2010-03-09 | 2010-08-11 | 珠海市英平生物科技有限公司 | Method for quickly amplifying target genes from genome DNA |
| 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 |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102676689B (en) * | 2012-06-11 | 2013-09-18 | 中国水产科学研究院黄海水产研究所 | Triple-PCR (Polymerase Chain Reaction) detection method for exopalaemon carinicauda by using microsatellite mark |
| CN105177158B (en) * | 2015-10-15 | 2018-04-03 | 天津商业大学 | A kind of Marsupenaeus japonicus micro-satellite triple PCR detection method |
-
2005
- 2005-01-25 CN CNB2005100424029A patent/CN100510103C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101798570A (en) * | 2010-03-09 | 2010-08-11 | 珠海市英平生物科技有限公司 | Method for quickly amplifying target genes from genome DNA |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1680601A (en) | 2005-10-12 |
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