Marine Invertebrates Dirk Steinke Short Course on DNA Barcoding Methods 29 Nov 2011
Preservation The good • Ethanol preserved, not older than 5 years • frozen tissue • RNA laterThe bad• Ethanol preserved, older than 10 years at room temp• DMSO (cross-reacts with Ethanol)• IsopropanolThe ugly• formalin• spirit•…
SamplingmtDNA rich tissue where possible• muscle tissue (larger animals e.g. fish)• legs (arthropods)• tube feet (echinoderms) The smaller the better!
ExtractionRegular kits will do in many cases, butEchinodermsPolychaetesMollusks work much better with CTAB extraction protocols (binds to polysaccharides that can inhibit PCR)
PCRTaxon-specific primers are key Check out http://connect.barcodeoflife.net/group/marinebarcoding
PCR Majority works with Taxon specific primersone primer pair or cocktail needed - Fish - Crustacea - Cephalopoda1 - Annelida - Gastropoda - Cnidaria - Pycnogonida - Porifera - Echinodermata - Brachipoda Notoriously difficult - Foraminifera - Bivalvia2 - Tunicata 1some families have a tandem copy of COI 2two different COI-versions (male/female)
SequencingMultiplex primer cocktails 5’ 3’ M13-tail • M13 tail for sequencing multiplex products • M13 also useful for standard primer pairs (Folmer-tailed version very successful)
Editing• some groups exhibit indels more frequently (e.g. crustacea, mollusks)• watch out for pseudogenes (often easy to spot through stop codons)• proper alignment is crucial• some symbiotic bacteria can be amplified using universal primers