Methodology Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA

Abstract

ABSTRACT. PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification.Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/µL) and concentration of PCR buffer (1X), MgCl 2 (2.5 mM) and dNTP mixture (200 µM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 µM primers for Cyt b and COI, 0.3 µM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4°C for Cyt b, 56.1°C for 12S and 51.3°C for COI. PCR products obtained under these conditions produced excellent DNA sequences.

Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies

“…Additionally, this gene has an evolutionary rate that is almost similar to that of mitochondrial DNA itself 17 . Gel analysis can give a broad classification of species by the size of the amplicons but is not as specific as sequencing the amplicon.…”

Molecular phylogenetic inference of White-Spotted Guitarfish (Rhynchobatus australiae) collected from local Malaysian fish markets

“…According to Troast et al (2016), applications of the COI locus alone failed to postulate population structures within species accurately. Thus, we propose that future studies at the population level should exploit other mtDNA gene sequences such as cytochrome b and D-loop regions that have been proven to analyze taxa at population levels (Rosli et al 2011;Ang et al 2012).…”

Expression of lymphocyte coding genes in peripheral blood and lymphocyte infiltration in cardiac tissues influenced by cyclosporin A in heterotopic heart transplantation model in rats

“…The expression of these genes occurred 24 h earlier than CD4 and CD8 surface protein expression, which suggested that they were upregulated as a downstream effect of T cells activation. 12S ribosomal takes part in cellular surface molecular translation and synthesis in the early stage of T cell activation [20][21][22]. MHC-III (hsc70t) is a participant in the cytotoxic cell activation process by coding complement C2, C3 and B factor [23,24].…”