Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays.
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/A280and 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.
Department of Biotechnology, Inter-University Centre for Genomics and Gene Technology, University of Kerala, Trivandrum, 695 581, India. lindakoshy@gmail.com.
Linda
L
Koshy
Department of Biotechnology, Inter-University Centre for Genomics and Gene Technology, University of Kerala, Trivandrum, 695 581, India.
A L
AL
Anju
Department of Cardiology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, 695 011, India.
S
S
Harikrishnan
Achutha Menon Centre for Health Science Studies, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, 695 011, India.
V R
VR
Kutty
Achutha Menon Centre for Health Science Studies, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, 695 011, India.
V T
VT
Jissa
Department of Biotechnology, Inter-University Centre for Genomics and Gene Technology, University of Kerala, Trivandrum, 695 581, India.
Irin
I
Kurikesu
Department of Biotechnology, Inter-University Centre for Genomics and Gene Technology, University of Kerala, Trivandrum, 695 581, India.
Parvathy
P
Jayachandran
Department of Biotechnology, Inter-University Centre for Genomics and Gene Technology, University of Kerala, Trivandrum, 695 581, India.
A
A
Jayakumaran Nair
Department of Biotechnology, Inter-University Centre for Genomics and Gene Technology, University of Kerala, Trivandrum, 695 581, India.|Deparment of Botany, University of Kerala, Trivandrum, 695 581, India.
A
A
Gangaprasad
Department of Biotechnology, Inter-University Centre for Genomics and Gene Technology, University of Kerala, Trivandrum, 695 581, India.
G M
GM
Nair
Department of Biotechnology, Inter-University Centre for Genomics and Gene Technology, University of Kerala, Trivandrum, 695 581, India.|Department of Computational Biology and Bioinformatics, University of Kerala, Trivandrum, 695 581, India.
P R
PR
Sudhakaran
D000328:Adult; D000818:Animals; D005591:Chemical Fractionation / Q000379:methods*; D003324:Coronary Artery Disease / Q000235:genetics*; D004247:DNA / Q000097:blood* / Q000302:isolation & purification / Q000592:standards*; D004272:DNA, Mitochondrial / Q000097:blood / Q000302:isolation & purification / Q000592:standards; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics; D014998:Y Chromosome / Q000235:genetics
D004272:DNA, Mitochondrial; D004247:DNA; C526097:Atp1a3 protein, mouse; D000254:Sodium-Potassium-Exchanging ATPase
DNA isolation; Purity; Real-time PCR; SNP genotyping; Whole blood
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