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

• Published in: Molecular biology reports 2017-02, Vol.44 (1), p.97-108
• Main Authors: 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.
• Format: Article
• Language: English
• Subjects: Adult; Animal Anatomy; Animal Biochemistry; Animals; Biomedical and Life Sciences; Cardiovascular disease; Chemical Fractionation - methods; Coronary Artery Disease - genetics; Deoxyribonucleic acid; DNA; DNA - blood; DNA - isolation & purification; DNA - standards; DNA, Mitochondrial - blood; DNA, Mitochondrial - isolation & purification; DNA, Mitochondrial - standards; Epidemiology; Extraction processes; Female; Genomics; Genotype & phenotype; Histology; Humans; Life Sciences; Male; Mice; Middle Aged; Molecular biology; Morphology; Original Article; Real-Time Polymerase Chain Reaction - methods; Sodium-Potassium-Exchanging ATPase - genetics; Y Chromosome - genetics
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/s11033-016-4085-9

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 (A 260 /A 280 and A 260 /A 230 ), 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.