ProtocolsResearchers have developed many protocols to expedite DNA barcoding, which are gaining usage by other members of the barcoding community. These protocols are useful for any lab, large or small, although recent work has focused on developing methods suitable for high-throughput barcode assembly (Hajibabaei et al. 2005). Further refinements to both lower the cost and boost productivity are a current priority. Additional work seeks to develop more reliable methods for recovering DNA from ancient samples.
DNA ExtractionExtraction DNA can be successfully recovered from a wide range of tissues. However, factors such as freshness, method of preservation, and age of the preserved specimen all have an impact on the overall success of obtaining a DNA sequence. These factors may influence i) the integrity of DNA within the specimen, as well as ii) the method of extraction.
Fresh, unpreserved tissue is ideal when conditions permit. However, many 'wet' tissues degrade rapidly once cell death occurs. Cell lysis and the release of enzymes can quickly destroy the DNA content of a sample. A general rule of thumb when using fresh material is 'the fresher, the better'!
Some common methods of preservation include flash-freezing, ethanol, dessication, and formalin. Flash-freezing from fresh tissue is a very effective means of preservation, and extraction results are typically comparable to using fresh tissue directly. Simple methods, such as a proteinase K digestion are usually sufficient. Ethanol is also a reliable method, where DNA may be readily recovered from specimens up to 10 years old using simple extraction techniques.
Dessicated or dried samples are often encountered when dealing with museum specimens. In some cases, simple methods may suffice, but more involved techniques may also be required (particulary with older samples). Formalin is another method common in older collections and presents a formidable challenge for DNA recovery. However, some protocols have been developed which have yielded modest amounts of DNA from formalinized tissue. Currently, two standard protocols developed at the CCDB, Guelph are available on this site - a simple proteinase K digestion for specimens in good condition, and an extraction kit (GenElute Mammalian Genomic DNA Miniprep Kit - Sigma Genosys) protocol for old or degraded specimens. For information on these protocols, please download our DNA Extraction document.
AmplificationThe amplification of target DNA is an important stage in current sequencing methods. DNA can be replicated using bacterial cultures (cloning), or via the polymerase chain reaction. PCR is often preferred due to the speed and high through-put of PCR systems. This is accomplished by placing the DNA extractions in a specialized chemical environment and cycling them through a specific temperature regime. A heat-activated DNA polymerase replicates the target DNA sequence with each cycle.
A critical component of the amplification process is the selection of appropriate primers. A list of currently used primers for DNA Barcoding is given here. Also PCR reagents, thermocycling profiles, primers, and general practices are available in PDF format .
Protocols for DNA visualization, purification, and sequencing reactions are available here.