Primer Optimization of the 13 Oxidative Phosphorylation Genes of Mitochondrial DNA

Introduction: Mitochondrial DNA (mtDNA) is a small circular genome comprising 37 genes of which 13 encode for oxidative phosphorylation. Due to its size and presence, isolation of pure mtDNA from biological samples is a challenge. In response, analysis of mtDNA from a mixed sample of genomic DNA and...

Full description

Saved in:
Bibliographic Details
Published inAsia-Pacific journal of molecular biology and biotechnology Vol. 30; p. 80
Main Authors Low, Hui Ching, Chilian, William M, Ratnam, Wickneswari, Ng, Zhi Xiang, Pung, Yuh-Fen
Format Journal Article
LanguageEnglish
Published Kuala Lumpur Malaysian Society for Molecular Biology and Biotechnology 01.06.2022
Subjects
Biological properties
Biological samples
Deoxyribonucleic acid
DNA
Efficiency
Genes
Leukocytes (mononuclear)
Mitochondrial DNA
Optimization
Oxidative phosphorylation
Peripheral blood mononuclear cells
Phosphorylation
Primers
Online AccessGet full text

Cover

More Information
Summary:Introduction: Mitochondrial DNA (mtDNA) is a small circular genome comprising 37 genes of which 13 encode for oxidative phosphorylation. Due to its size and presence, isolation of pure mtDNA from biological samples is a challenge. In response, analysis of mtDNA from a mixed sample of genomic DNA and mtDNA has been suggested. The objective of this study is to validate the presence of mtDNA in an isolated sample of total DNA and to determine the efficiency of the 13 oxidative phosphorylation (OXPHOS) primers. Methods: Using total DNA isolated from peripheral blood mononuclear cells (PBMCs) and minor amendments to the KAPA SYBR Fast Universal kit protocol, gradient PCR was run to obtain the annealing temperature (Ta) of the primers. Then, triplicates of five-point 10-fold dilutions (10 ng/μl to 0.001 ng/μl) were performed for the generation of the standard curves. The efficiency of each primer set was calculated from the standard curve. Results: The lowest quantification cycles (Cq) of the primers ranged from 19 to 21 at Ta of 54oC to 58oC. However, the lowest Cq value of ND2 was 24 at Ta of 56oC. For 0.1 ng/μl to 10 ng/μl of DNA, the Cq values were within the range of 20 to 30 while lower DNA input resulted in Cq values above 30. The primer efficiency ranged from 88 to 97%. Conclusion: The ideal Ta of the primers ranges from 54oC to 58oC. Results of the standard curve indicated 0.1 ng/μl to 10 ng/μl are the ideal amount of input DNA for amplification of each primer set.
ISSN:0128-7451