Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects

Abstract

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation.

A hands-on overview of tissue preservation methods for molecular genetic analyses

“…Gilbert et al 2007;Hunter et al 2008;Phillips and Simon 1995;Pichler et al 2001;Rohland et al 2004;Rowley et al 2007;Wisely et al 2004).…”

Four years of DNA barcoding: Current advances and prospects

“…Indeed, new techniques of non-destructive DNA extraction from recently collected specimens have already been developed (Pook and McEwing, 2005;Hunter et al, 2007;Rowley et al, 2007) and additional improvements in specimen conservation may arise. One major drawback of molecularbased studies as for example DNA barcoding is our incapacity to extract DNA from specimens conserved in formalin.…”

A simple and cost-effective protocol for extraction of genomic DNA from ethanol preserved black flies (Simuliidae: Diptera)

“…While many other high-throughput extraction methods published so far can offer similar or better efficiency, and they usually rely on some type of non-standard equipment, like sonicators, mills or robots [16][17][18][19][20][21][22]. In other cases, expensive deep-well plates are required.…”