Abstract
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.
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Acknowledgement
LK acknowledges the Science and Engineering Research Board-Department of Science and Technology (SERB-DST), Govt. of India, New Delhi, for financial support via the Fast Track Young Scientists Scheme, Project File No. SB/YS/LS-174/2013. We thank Dr. Mahesh S. K. for kindly providing mouse cDNA.
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The study protocol was approved by the Institutional Ethics Committee for Clinical Studies at the SCTIMST, IEC No. SCT/IEC/558/JUNE-2014.
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Koshy, L., Anju, A.L., Harikrishnan, S. et al. Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays. Mol Biol Rep 44, 97–108 (2017). https://doi.org/10.1007/s11033-016-4085-9
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DOI: https://doi.org/10.1007/s11033-016-4085-9