Marine InvertebratesDirk Steinke

Short Course on DNA Barcoding Methods 29 Nov 2011

Preservation

The good• Ethanol preserved, not older than 5 years• frozen tissue• RNA later

The bad• Ethanol preserved, older than 10 years at room temp• DMSO (cross-reacts with Ethanol)• Isopropanol

The ugly• formalin• spirit• …

Sampling

mtDNA rich tissue where possible

• muscle tissue (larger animals e.g. fish)• legs (arthropods)• tube feet (echinoderms)

The smaller the better!

Extraction

Regular kits will do in many cases, but

EchinodermsPolychaetesMollusks

work much better with CTAB extraction protocols(binds to polysaccharides that can inhibit PCR)

PCR

Taxon-specific primers are key

Check out http://connect.barcodeoflife.net/group/marinebarcoding

PCR

Majority works withone primer pair or cocktail

Taxon specific primersneeded

- Fish- Cephalopoda1

- Gastropoda- Pycnogonida- Echinodermata- Brachipoda

1some families have a tandem copy of COI2two different COI-versions (male/female)

- Crustacea- Annelida- Cnidaria- Porifera

Notoriously difficult

- Foraminifera- Bivalvia2

- Tunicata

Sequencing

Multiplex primer cocktails

M13-tail

5’ 3’

• 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

Thank you!