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United States Department of Agriculture

Agricultural Research Service

Title: A Simple and Effective Procedure for Discovering Microsatellites from Small Insert Libraries)

Author
item Wang, Xinwang
item Trigiano, Robert
item Windham, Mark
item Devries, Renae
item Scheffler, Brian
item Rinehart, Timothy
item Spiers, James

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.

Last Modified: 8/24/2016
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