A Microsatellite Marker, Specific Primer and Application for Identification of Silver Carp and Bighead Carp Hybrid and Genetic Introgression Individuals
A microsatellite marker and microsatellite technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem that it is difficult to accurately determine whether the tested individual is interspecies hybrid or genetically introgressed individual, etc. problems, to achieve the effect of reliable detection results, fast detection speed and high resolution
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Embodiment 1
[0032] [Example 1] Screening of microsatellite sites
[0033] The 18 silver carp and bighead carp interspecies specific microsatellite markers in the present invention are obtained through three stringent screenings. In the previous research of our laboratory, the microsatellite enrichment library of silver carp and bighead carp was constructed by magnetic bead enrichment method, and the sequences containing microsatellite repeats were obtained by cloning and sequencing. From the sequences containing microsatellites, select the sequences whose core repeats more than 5 times and meet the requirements of primer design, and use Primer Premier5.0 to design primers. The main parameters are set as follows: primer length 20-25bp, PCR product fragment length range 120-350bp, optimum annealing temperature 50-60°C. The GC content is generally between 40% and 60%, and the secondary structure should be avoided as much as possible. Finally, 227 microsatellite markers of silver carp and b...
Embodiment 2
[0037] [Example 2] Composition and preparation of silver carp and bighead carp interspecific microsatellite marker identification PCR system and electrophoresis detection amplification product system
[0038] A).PCR system composition:
[0039] dNTPs (10mM) are products of Shanghai Sangon Company; Taq DNA polymerase (5U / μL), 10×PCRBuffer are products of Dalian TaKaRa Company;
[0040] B). Composition of 10×PCR Buffer:
[0041] Tris-HCl (pH8.3) 100mM, KCl500mM, MgCl215mM;
[0042] C). PCR reaction system preparation:
[0043] The total volume is 12.5 μL, including 20-50ng template DNA, 0.4U Taq DNA polymerase, 1.25 μL 10×PCR Buffer, 0.4 μL dNTP (2.5mM), 0.4 μL forward and reverse mixed primers (2.5 μM each), and finally add appropriate amount Sterilize ultrapure water to 12.5 μL;
[0044] D). Polyacrylamide gel preparation:
[0045] 30% acrylamide 8.5mL, 5×TBE 5mL, 10% ammonium persulfate 0.5mL, TEMED 5μL, distilled water 11.5mL.
Embodiment 3
[0046] [Example 3] Species-specific microsatellite marker identification of silver carp and bighead carp and identification of silver carp, bighead carp and their hybrid individuals morphologically judged
[0047] A). Take three individuals to be identified as silver carp, bighead carp and hybrid according to their morphological characteristics, use the phenol / chloroform method to extract the genomic DNA of each individual, and dilute the DNA to a concentration of 20-50ng / μL.
[0048] B). Using the DNA extracted in step A) as a template, prepare a PCR reaction mixture according to the system in step C) of Example 2. It is necessary to prepare a PCR reaction mixture using the identified purebred silver carp and bighead carp individual DNA as a template as a control.
[0049] C). Amplify on a PCR machine. After pre-denaturation at 94°C for 5 minutes, perform 35 PCR reaction cycles. Each cycle includes denaturation at 94°C for 35 seconds, annealing at an appropriate annealing tem...
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