title: 'Direct PCR' optimization yields a rapid, cost-effective, nondestructive and efficient method for obtaining DNA barcodes without DNA extraction.
abstract: Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction ('direct PCR') and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore.
authors.forename: Wing Hing
authors.initials: WH
authors.lastname: Wong
authors.forename: Ywee Chieh
authors.initials: YC
authors.lastname: Tay
authors.forename: Jayanthi
authors.initials: J
authors.lastname: Puniamoorthy
authors.forename: Michael
authors.initials: M
authors.lastname: Balke
authors.forename: Peter S
authors.initials: PS
authors.lastname: Cranston
authors.forename: Rudolf
authors.initials: R
authors.lastname: Meier
mesh_terms: D000818:Animals; D002683:Chironomidae / Q000145:classification* / Q000235:genetics*; D003365:Costs and Cost Analysis; D058893:DNA Barcoding, Taxonomic / Q000191:economics / Q000379:methods*; D007814:Larva / Q000145:classification / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D017422:Sequence Analysis, DNA; D013997:Time Factors
keywords: Chironomidae; DNA barcodes; DNA extraction; macroinvertebrates