Using microwaves to prepare gastropods for DNA barcoding.
Extracting DNA from gastropods presents particular difficulties due to the capacity of the living animal to retract into the shell, resulting in poor penetration of the ethanol into the tissues. Because the shell is essential to establish the link between sequences and traditional taxonomic identity, cracking the shell to facilitate fixation is not ideal. Several methods are currently in routine use to overcome this difficulty, including chemical relaxation, drilling the shell and boiling. Most of these methods are time-consuming, may be safety hazards and constitute a bottleneck in the preparation of large numbers of specimens in the field. We have experimented with a method traditionally used to clean shells that involves placing the living gastropods in a microwave (MW) oven; the electromagnetic radiation very quickly heats both the animal and the water trapped inside the shell, resulting in separation of the muscles that anchor the animal to the shell. Done properly, the body can be removed intact from the shell and the shell voucher is preserved undamaged. To test the method, the bodies of live-collected specimens from two gastropod species were separated from their shell by microwaving and by anesthetizing/drilling. After identical extraction and PCR procedures, the gels showed no difference in DNA quantity or quality, and the resulting sequences are identical within species. The method was then implemented on a large scale during expeditions, resulting in higher percentage of DNA extraction success. The MWs are also effective for quickly and easily removing other molluscs from their shells, that is, bivalves and scaphopods. Workflows implementing the MW technique show a three- to fivefold increase in productivity compared with other methods.
Muséum National d'Histoire Naturelle, Département Systématique et Evolution, ISyEB Institut (UMR 7205 CNRS/UPMC/MNHN/EPHE), 43, Rue Cuvier, 75231, Paris, France.
L A
LA
Galindo
N
N
Puillandre
E E
EE
Strong
P
P
Bouchet
D000818:Animals; D004247:DNA / Q000302:isolation & purification*; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D049851:Gastropoda / Q000145:classification / Q000235:genetics*; D008872:Microwaves; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA
D004247:DNA
DNA damage; PCR optimization; shelled molluscs; tissue preparation; voucher