A simplified arthropod genomic-DNA extraction protocol for polymerase chain reaction (PCR)-based specimen identification through barcoding |
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| Authors: | Venu M Margam Emma W Gachomo John H Shukle Oluwole O Ariyo Manfredo J Seufferheld Simeon O Kotchoni |
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| Institution: | (1) Department of Entomology, Purdue University, Smith Hall, 901 West State Street, West Lafayette, IN 47907, USA;(2) Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USA;(3) Department of Mathematics and Natural Sciences, Allen University, Columbia, SC 29204, USA;(4) Department of Crop Science, University of Illinois at Urbana-Champaign, 311 ERML, 1201 W Gregory Drive, Urbana, IL 61801, USA;(5) Department of Agronomy, Purdue University, Lilly Hall, 915 West State Street, West Lafayette, IN 47907, USA; |
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| Abstract: | Genomic DNA extraction protocols generally require the use of expensive and hazardous reagents necessary for decontamination
of phenolic compounds from the extracts. In addition, they are lengthy, hindering large-scale sample extractions necessary
for high-throughput analyses. Here we describe a simple, time and cost-efficient method for genomic DNA extraction from insects.
The extracted DNA was successfully used in a Polymerase Chain Reaction (PCR), making it suitable for automation for large-scale
genetic analysis and barcoding studies. The protocol employs a single purification step to remove polysaccharides and other
contaminating compounds using a non-hazardous reagent buffer. In addition, we conducted a bioinformatics database analysis
as proof of concept for the efficiency of the DNA extraction protocol by using universal barcoding primers specific for cytochrome
c oxidase I gene to identify different arthropod specimens through Barcode of Life Database (BOLD) database search. The usefulness
of this protocol in various molecular biology and biodiversity studies is further discussed. |
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