Overview DNA barcoding demystified Andrew Mitchell* NSW Department of Primary Industries, Wagga WaggaAgricultural Institute, Private Mail Bag, Wagga Wagga, NSW 2650, Australia. Key words barcode, DNA taxonomy, molecular diagnostics. INTRODUCTION Only 10% of the earth’s biota has been described despite 250 years of taxonomic research (Wilson 2000). This is in large part a reflection of the extent and complexity of biological diversity, but it is also true that traditional taxonomic techniques are labourious and highly specialised, and taxonomic expertise is very thinly spread across the myriad groups of life (Scotland et al. 2003). As a result, obtaining species identifications, even for a modest sample of commonly encountered insects in a biologically diverse country, such as Australia, is at best time- consuming and costly and all too often impossible. This is a result of the difficulty that morphological taxonomic techniques encounter in dealing with phenotypic differences between immature and mature stages of a species, and among individu- als of variable species, the lack of phenotypic differences among cryptic species, shifts in geographical distribution as a result of invasions or range expansions and the overwhelming diversity of life. Our reliance on morphological approaches, thus, places serious limitations on the diagnosis of biological diversity at a time when we are facing a biodiversity crisis of unprecedented magnitude. It has long been clear that new approaches to biodiversity assessment, description and diagno- sis are needed, and indeed, the 1990s saw the emergence of many novel taxonomic initiatives, leading E.O. Wilson to con- clude that ‘the 19th century culture of taxonomy has begun to be replaced’ (Wilson 2003). The emerging discipline of DNA barcoding (Floyd et al. 2002; Hebert et al. 2003) represents another advance and part of the solution to these problems. This paper provides a brief overview of DNA barcoding, and con- siders what barcoding offers researchers and industry alike. WHAT ARE DNA BARCODES AND ARE THEY NOVEL? DNA barcodes are short DNA sequences (500 base pairs) that can be used to identify species. The ‘barcode’ analogy refers, of course to the universal product codes (UPC) affixed to merchandise in retail stores. The analogy suggests that DNA barcodes similarly provide a universal system of readily scan- nable, unique tags for products (species), speeding and auto- mating transactions (identifications) and facilitating novel applications, such as automated stock management (biodiver- sity assessment) and so on. Unfortunately, the barcode analogy also has been used by the microbial diagnostics community to refer to the banding pattern of rep-polymerase chain reaction (PCR) products (a technique similar in principle to Random Amplification of Polymorphic DNA (RAPD)-PCR) visualised on a gel, which bears some resemblance to UPCs (Sutton & Cundell 2004). Love it or hate it, the term ‘DNA barcoding’ has caught the public imagination, and is likely here to stay. DNA sequence data from the mitochondrial genome have long been used to resolve difficult taxonomic questions and diagnose species in cases where morphology alone has proved inadequate (e.g. Brown et al. 1999; Mitchell & Samways 2005), so is DNA barcoding just molecular systematics and molecular diagnostics repackaged or is it a truly novel enter- prise? The novelty in DNA barcoding is its combination of four main factors: standardisation on a particular gene region, the large scale of operation, compulsory vouchering of speci- mens and active curation of the resulting databases. First, standardisation on a particular gene region for all species in a particular branch of life leads to universality of the system. For animals the standard is the upstream half of the mitochondrial gene cytochrome c oxidase subunit I (COI or cox1). Without standardisation, molecular systematic data sets lack interoperability, building ‘Towers of Babel’ (Caterino et al. 2000). The same is true of molecular diagnostics – the literature contains a plethora of highly specialised methods for identifying particular organisms, and this leads to some circu- larity in that one has to have a well-developed idea of an organism’s identity (e.g. which genus) to even know which diagnostic test to apply. Furthermore, non-sequence-based diagnostic tests are incapable of uniquely identifying novel isolates, and must be redeveloped if novel isolates are discov- ered. Standardisation allows for the generalisation of the pro- tocols and skills needed to implement diagnostics so that identifications can be performed by technicians with generic molecular biology skills. Second, the use of high-throughput techniques adapted from genomics allows small molecular biology laboratories to process hundreds of samples per day (thousands if they use *andrew.mitchell@dpi.nsw.gov.au Australian Journal of Entomology (2008) 47, 169–173 © 2008 The Author Journal compilation © 2008 Australian Entomological Society doi:10.1111/j.1440-6055.2008.00645.x