Submitted to: Marine Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 1, 2005
Publication Date: May 25, 2006
Citation: Rodriguez, M.F., Gahr, S.A., Rexroad III, C.E., Palti, Y. 2006. A pcr screening method for rapid detection of microsatellites in bacterial artificial chromosomes (bacs). Marine Biotechnology DOI: 10.1007/s10126-005-5064-7. Online Publication 5/25/2006. Interpretive Summary: Microsatellites are often the marker system of choice for genetic mapping due to their high degrees of polymorphism, co-dominant inheritance and genome wide distribution. Genome research is facilitated by species-specific tools such as large insert genomic library of bacterial artificial chromosomes (BACs).Our goals in developing microsatellites from BACs are to map neighboring genes contained in the same BACs and to integrate physical and genetic maps. In this report we describe an alternative non-radioactive PCR based method for rapid identification of microsatellites in rainbow trout BAC clones, which may be useful for similar projects involving other species.
Technical Abstract: Standard protocols aimed at identifying sub-clones of interest from bacterial artificial chromosomes (BACs) include the use of hybridization methods which are time consuming and often require the use of radioactive isotopes. Through our efforts to identify microsatellites in BACs from rainbow trout (Oncorhynchus mykiss) we have developed a non-radioactive PCR-based screening technique to select microsatellite containing sub-clones for marker development. Two BACs were sub-cloned and screened by PCR using a vector specific primer and a mix of microsatellite repeat primers. The sub-clones were then sequenced to evaluate the efficiency of the PCR screening method. Correlation between positive PCR amplification and presence of microsatellites varied between the 2 BACs (21.9 % and 71.4 %), but still sufficient number of sub-clones were identified to enable design and optimization of microsatellite markers.