A simplified arthropod genomic-DNA extraction protocol for polymerase chain reaction (PCR)-based specimen identification through barcoding.
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.
Margam
Venu M
VM
Department of Entomology, Purdue University, Smith Hall, 901 West State Street, West Lafayette, IN 47907, USA.
Gachomo
Emma W
EW
Shukle
John H
JH
Ariyo
Oluwole O
OO
Seufferheld
Manfredo J
MJ
Kotchoni
Simeon O
SO
D000818:Animals; D001181:Arthropods / Q000145:classification* / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D016678:Genome / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004247:DNA