Main content area

Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays

Koshy, Linda, Anju, A. L., Harikrishnan, S., Kutty, V. R., Jissa, V. T., Kurikesu, Irin, Jayachandran, Parvathy, Jayakumaran Nair, A., Gangaprasad, A., Nair, G. M., Sudhakaran, P. R.
Molecular biology reports 2017 v.44 no.1 pp. 97-108
DNA, agar gel electrophoresis, blood, coronary artery disease, cost effectiveness, epidemiological studies, genotyping, humans, isolation techniques, mice, patients, quantitative polymerase chain reaction, restriction fragment length polymorphism, silica, single nucleotide polymorphism
The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol–chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol–chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.