Tối ưu hoá điều kiện PCR trong khuếch đại đoạn Intron 2 của gen leptin và vùng 5’Utr của gen thyroglobulin trên DNA tách chiết từ gốc lông bò

Abstract

The intron 2 of the leptin gene (LEP) and the 5′ untranslated region (5' UTR) of the thyroglobulin gene (TG5) are considered candidate genes for selecting traits related to beef yield and quality. This study was conducted to optimize the PCR conditions required to amplify the LEP and TG5 genes. The AccuRive sDNA/RNA Prep kit was used to extract genomic DNA from hair roots of cattle. The reaction mixture of 20 μL final volume contained 1.25 µM primers, 200 mM dNTP, 1× PCR buffer, 0.75 unit Taq polymerase, and genomic DNA. PCR is optimized with the determination of primer annealing temperature and template DNA concentration. The investigated annealing temperature range was 55-64°C for the LEPF/LEPR primers and 52-58°C for the TGF/TGR primers. The optimal concentration of template DNA was tested in the range of 25-150 ng. The results showed that the suitable annealing temperature range for LEPF/LEPR and TGF/TGR primer pairs were 58-62°C and 52-58^oC, respectively. The optimal annealing temperature to amplify the LEP and TG5 genes was 62°C and 55°C. The genomic DNA concentration of about 50–75 ng was essential to achieve this amplification in the case using 1.25 µM of primers. The multiplex PCR for LEP and TG5 genes can be implemented with annealing temperature of 58^oC.