Optimization and Validation of PCR protocol for three Hypervariable Regions (HVI, HVII and HVIII) in Human Mitochondrial DNA

Shakeela Daud, Saqib Shahzad, Muhammad Shafique, Munir Ahmad Bhinder, Muhammad Niaz, Asif Naeem, Zia ur Rehman, Tayyab Husnain

Abstract


Background: Due to high copy number of mitochondria per cell mtDNA testing is often successful in cases where nuclear DNA is highly degraded or the sample source is too limited. Sequence polymorphism of D-loop region of mtDNA has been used for identification of forensic remains, analysis of mother–child relationships and comparisons between ethnic groups through maternal lineages. PCR conditions were optimized and validated for three hypervariable regions (HVR I- 480 bp, HVR II- 420 bp and HVIII- 255 bp) of mitochondria conducted at National Centre of Excellence in Molecular Biology, University of the Punjab - Lahore, Pakistan.

Methodology: Blood samples of 86 individuals were drawn from 25 Pakistani families. DNA was extracted and purified by Sambrook method. DNA was quantified at agarose gel electrophoresis and N-D 1000 Nanodrop spectrophotometer. Three hypervariable regions (HVR I, HVR II and HVIII) of mitochondrial DNA were optimized with different PCR components and PCR conditions using three pairs of oligonucleotides along with reagent blanks, positive and negative controls.

Results: Three hypervariable regions (HVR I, HVR II and HVIII) of mitochondrial DNA were optimized in the PCR reaction volume at different concentrations of PCR buffer, MgCl2, Taq DNA polymerase, dNTPs, primers and mtDNA template at varying annealing temperatures. The best results for amplification were shown at 1x PCR buffer, 2.5mM Mgcl2, 0.3µl of Taq DNA polymerase (5u/µl), 0.2mM dNTPs, 0.8µM forward-reverse primers for HVR I, 0.7µM forward-reverse primers for HVR II and 0.4µM forward-reverse primers for HVR III at 52◦C annealing temperature.

Conclusion: Optimized PCR protocol for three hypervariable mtDNA regions has provided a way out to lead mtDNA analysis which is very necessary tool in those forensic biological samples, where nuclear DNA is highly degraded, to identify missing persons and determine maternal lineages.


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