|Rinehart, Timothy - Tim|
Submitted to: Molecular Ecology Notes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/6/2006
Publication Date: 12/14/2006
Citation: Wang, X., Trigiano, R.N., Windham, M.T., Devries, R., Scheffler, B.E., Rinehart, T.A., Spiers, J.M. 2006. A simple and effective procedure for discovering microsatellites from small insert libraries. Molecular Ecology Notes. Vol 7. pp 558-561
Interpretive Summary: Here we describe a rapid, cost effective procedure to screen colonies for the correct inserts using a simple PCR reaction with three primers to expose microsatellite repeats. We confirm that of 700 colonies from 6 libraries, 96-100% contained the desired microsatellite motif using our new procedure. This procedure may be applied to any microsatellite-enriched libraries as well as small insert genomic libraries. We believe cost savings could be 40% over traditional method of sequencing all the clones.
Technical Abstract: Microsatellite loci are useful markers for studying genetic diversity and for creating linkage maps in plants and animals. However, microsatellite discovery from a genomic library is tedious and costly. We have constructed a small insert genomic library and six repeats-enriched libraries. Instead of using colony hybridization, we used a simple PCR reaction with three primers to directly amplify the insert to expose microsatellite repeats from both genomic and repeat-enriched libraries. Sequencing results showed that those colonies, which PCR amplifications revealed a long strong smear, plus sometimes a band in agarose gel, contained the desired microsatellite motif whereas, a single strong band in a lane indicated the lack of an SSR. Approximately 700 of the former colonies were selected from 6 libraries, the insert sequenced and 96-100% contained the desired microsatellite motif. This simple PCR method to discover microsatellite directly from insert-containing colony is efficient and cost effective way to identify SSR containing colonies. This procedure may be applied to any microsatellite-enriched libraries as well as small insert genomic libraries. For microsatellite-enriched libraries we believe minimal cost savings could be 40% over traditional method of sequencing all the clones.