05 January 2007

Nuclear gene development for FLYTREE AToL

Brian Wiegmann, Norman Barr, and Jungwook Kim have been working on nuclear gene development for the the FLYTREE AToL project (Diptera).

The following is a list of the genes currently being using in FLYTREE:
AATS (~3k)
CAD (~4k)
g6pd (~1k)
GART (~1.4k)
pepck (~700bp)
per (~700bp)
pgd (~800bp)
pug (~1.5k)
sia (~450bp)
snf (~400bp)
stx (~600bp)
tpi (~1k)

Our lab strategies are very similar to (and often based on...) the Regier lab protocols; however, we primarily use genomic DNA as a starting template, and only use RT PCR for individual freshly preserved taxa that have proven difficult to amplify. This often means multiple rounds of taxon specific primer design and testing.

We are developing and testing primers for two additional genes: pug and stx.

We have tried a number of other genes in the Regier lab list without much success. In the above list, GART is very difficult to work with because it has unpredictable introns, unpredictable AA sequence variation, and a large internal repeat. We divide AATS and CAD into smaller sized fragments for amplification. These segments of AATS and CAD provide differing levels of success in different taxa, and so can also be challenging.

Jungwook Kim, Ph. D.
FLYTREE postdoc
Department of Entomology
North Carolina State University
Raleigh, NC 27695