Microsatellite Marker Primer Combination and Its Application for Phylogenetic Analysis of Yellow-cheeked Gibbon
A technology of microsatellite markers and primer combinations, applied in the field of molecular genetics, can solve the problems of ambiguous kinship and inaccurate genealogy, and achieve the effects of accurate kinship analysis data, high polymorphism, and cost and time savings
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Embodiment 1
[0038] 1. Genomic DNA Extraction
[0039] Take 20 yellow-cheeked gibbon hairs with hair follicles and place them in 2ml centrifuge tubes, add 600 μL of cell lysate (Tris-HCl 100mM, pH 8.0; EDTA 50mm / L, pH 8.0; SDS1%, pH 8.0; NaCl 125mM ), add 9 μL of proteinase K at a concentration of 20 mg / mL to each tube, place it in a 60°C water bath for 2-4 hours, and shake the centrifuge tube every half hour until the tissue is fully lysed. Cool the centrifuge tube to room temperature, add 200 μL of 7.5M ammonium acetate, shake well, place in a refrigerator at 4°C for 5 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, and transfer 500ml of the supernatant to a new 1.5ml centrifuge tube. Add 600ml of isopropanol, mix well, precipitate at room temperature for 1-2min, centrifuge at 12000rpm at 4°C for 10min, and discard the isopropanol. Add 70% ethanol to wash the DNA, centrifuge at 12000rpm at 4°C for 5min, and discard the 70% ethanol. Add absolute ethanol, centrifuge at 12000 rpm...
Embodiment 2
[0049] 1. Experimental objects and methods
[0050] The primers of the 11 microsatellite sites screened in Example 1 were used to analyze the genetic relationship of 14 yellow-cheeked gibbons that were identified as yellow-cheeked gibbons through species identification.
[0051]The hair of the yellow-cheeked gibbon was collected, and the genomic DNA in the hair follicle of the gibbon was extracted according to the genome extraction method in Example 1. The extracted genomic DNA samples were stored at -20°C for future use. Using the extracted genomic DNA as a template, PCR was carried out with the above 11 pairs of primers; the amplified products were typed on the ABI3730XL Genetic Analyzer using the above 11 pairs of primers, and the GS-500 was used as an internal reference and read by GeneMarkerV1.5 software The individual's genotype. The software CERVUS 3.0 was used to calculate the allele frequency, heterozygosity, expected heterozygosity, polymorphic information content,...
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