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

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• 作者：Linda Koshy ; A. L. Anju ; S. Harikrishnan ; V. R. Kutty…
• 关键词：Whole blood ; DNA isolation ; Purity ; SNP genotyping ; Real ; time PCR
• 刊名：Molecular Biology Reports
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：44
• 期：1
• 页码：97-108
• 全文大小：
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Animal Biochemistry; Animal Anatomy / Morphology / Histology;
• 出版者：Springer Netherlands
• ISSN：1573-4978
• 卷排序：44

文摘

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.