SELECTION OF HYBRIDS BY AFFINITY CAPTURE (SHAC) - A METHOD FOR THE GENERATION OF CDNAS ENRICHED IN SEQUENCES FROM A SPECIFIC CHROMOSOME REGION

We have established a method for preparing cDNA sublibraries enriched in sequences from specific chromosome regions, called selection of hyb rids by affinity capture (SHAC). This procedure can be described in tw o stages. In the first stage, a particular chromosome region, in this study mouse chromosome 11, was microdissected, followed by PCR amplifi cation with a universal degenerate primer. This material is referred t o as the ''target'' DNA. In the second stage, a mouse liver cDNA libra ry with unique linker-adapter ends, referred to as the ''source'' cDNA , was hybridized to the biotin-labeled target DNA prepared during the first stage. The resulting DNA duplexes were captured by streptavidin- coated magnetic beads. The cDNAs were released from their biotin-label ed target homologs by alkaline denaturation and recovered by PCR ampli fication. These cDNAs were referred to as the SHACcDNAs. Specificity o f the SHACcDNA to chromosome 11 was verified by FISH analysis. To exam ine representation of the SHACcDNA, we confirmed the presence of seven genes or single-copy DNA segments known to be localized on mouse chro mosome 11, using a dot blot assay. In addition, a second round of SHAC was performed to achieve even higher specificity for the resulting ch romosome 11 SHACcDNA. The SHAC technology should facilitate constructi on of cytogenetically defined cDNA libraries and should assist in the fields of gene discovery and genome mapping. (C) 1995 Academic Press, Inc.