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Sextuple PCR (polymerase chain reaction) detection method of portunus trituberculatus miers microsatellite marker

A technology of Portunus trituratus and microsatellite markers, which is applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as time-consuming, complicated operation, etc., and achieve the effects of improving efficiency, shortening experimental time, and being easy to detect.

Active Publication Date: 2011-08-17
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Abstract
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  • Application Information

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Problems solved by technology

[0005] Aiming at the shortcomings of complicated operation and long time-consuming existing in the traditional breeding of Portunus trituberculatus in the prior art, the present invention provides a six-fold PCR detection method of microsatellite markers in Portunus trituberculatus. On the basis of microsatellite markers, the combination and collocation of markers and the optimization of experimental methods are carried out to obtain a simpler and faster detection method, which greatly shortens the experimental time. Compared with the conventional PCR method, the efficiency is increased by about 6 times, and the obtained results can reflect the Genetic Variation of Multiple Microsatellite Loci in Portunus trituberculatus

Method used

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  • Sextuple PCR (polymerase chain reaction) detection method of portunus trituberculatus miers microsatellite marker
  • Sextuple PCR (polymerase chain reaction) detection method of portunus trituberculatus miers microsatellite marker
  • Sextuple PCR (polymerase chain reaction) detection method of portunus trituberculatus miers microsatellite marker

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Embodiment 1

[0032] A six-fold PCR construction method of Portunus trituberculatus microsatellite markers of the present invention is as follows:

[0033] 1. Based on the microsatellite core sequence in the Portunus trituberculatus microsatellite enrichment library provided by the present invention, use Primer Premier 6.0 software to design corresponding PCR primers at the two ends of its microsatellite core sequence to obtain a series of PCR primers with PCR amplification. Amplified SSR markers of varying product sizes.

[0034] 2. Use Primer Premier 6.0 software to evaluate between primers, and select the primers that show most of the ΔG (Gibbs of energy change) value above -4 for use, and the ΔG (Gibbs of energy change) value above -4 is mainly explained In the multiplex PCR system, it is difficult for a single pair of primers to form primer dimers and have a low hairpin structure, so as not to affect the efficiency of the PCR reaction. Through the matching and combination of 22 pairs ...

Embodiment 2

[0049] This embodiment is aimed at Mg 2+ Concentration optimization experiment was carried out, the experimental process is as follows:

[0050] Steps (1) and (2) are the same as in Example 1.

[0051] The PCR reaction system in step (3) is: 10×Buffer 1.5 μ L; 2.5 mmol / LdNTP 1.5 μ L; Ptr33a of the present invention, Ptr95, Pot42, Pot44 and Pot57 primers are respectively 0.4 μ L, Pot34 positive and negative primers respectively 0.5 μ L, each Primer concentration is 10pmol / μL; 50ng / μL Portunus trituberculatus genomic DNA 0.6μL; 5U / μL Taq polymerase 0.12μL; ddH 2 O supplemented to 15 μL; 15 mmol / L MgCl 2 The volume was optimized with a gradient of 0.8, 1.0, 1.2, 1.4 μL.

[0052] The reaction program used the conventional PCR reaction program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 40 s, annealing at 60°C for 1 min, extension at 72°C for 1 min, and a total of 25 cycles. Finally, extend at 72°C for 5 minutes, store at 4°C and end the program.

[0053] The...

Embodiment 3

[0055] This embodiment selects the above-mentioned optimized reaction program and Mg 2+ Volume, the six-fold PCR detection method concrete steps of a kind of Portunus trituberculatus microsatellite marker of the present invention are as follows:

[0056] (1) The selected six primer sequences of the present invention are shown in Table 2 below:

[0057] Table 2: Six primer sequences and their annealing temperatures and accession numbers of their core sequences

[0058]

[0059] (2) Genomic DNA extraction of Portunus trituberculatus: the large claw muscle tissue of Portunus trituberculatus was taken, and the genomic DNA of Portunus trituberculatus was extracted by the method of phenol / chloroform. The integrity of genomic DNA was detected by 1% agarose gel electrophoresis, the concentration of genomic DNA was detected by RNA / DNA spectrophotometer and diluted to a final concentration of 50ng / μl, and stored at -20°C for later use.

[0060] (3), PCR reaction system: 10×Buffer 1...

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Abstract

The invention provides a sextuple PCR (polymerase chain reaction) detection method of a portunus trituberculatus miers microsatellite marker. The detection method comprises the following steps of: designing and synthesizing a sextuple PCR primer; extracting genome DNA (deoxyribonucleic acid); performing sextuple PCR reaction; and detecting a PCR product, wherein the sextuple PCR reaction comprises a sextuple PCR reaction system and a sextuple PCR reaction procedure. The method builds the sextuple PCR reaction system which performs the portunus trituberculatus miers family and paternity test to simultaneously detect six microsatellite sites in the PCR reaction, so that efficiency is improved by about six times compared with the existing PCR method. The method has the characteristics of being high-efficiency, economical, simple, convenient and the like, thereby being capable of being popularized and applied in the portunus trituberculatus miers genetic diversity analysis, the genetic relationship test, the family management and the improved variety breeding.

Description

technical field [0001] The invention relates to a PCR detection method for microsatellite markers of Portunus trituberculatus, in particular to a six-fold PCR detection method for microsatellite markers of Portunus trituberculatus. Background technique [0002] Microsatellites (microsatellites) or simple sequence repeats (simple sequence repeats, SSR) refers to a simple tandem repeat DNA sequence consisting of 1 to 6 nucleotides. It has been found in all biological species studied so far, and its distribution density is very large. Because microsatellites are widely distributed in the genome, they have the characteristics of high density, rich polymorphism, high heterozygosity and good stability, co-dominant inheritance following Mendelian segregation law, and easy PCR amplification. It is an eye-catching new type of DNA marker, and is widely used in many research fields such as family genealogy authentication of biological resources, gene linkage analysis, genetic map cons...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 刘萍
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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