This proposal describes a method for preparing cDNA sublibraries specific to certain chromosome regions. The procedure, which we call selection of hybrids by affinity capture (SHAC), can be described in two stages. In the first stage, banded mitotic chromosomes are microdissected and PCR- amplified using a universal degenerate primer. This is a general technique employed in our lab to obtain chromosome region-specific genomic DNA; the specificity of the resulting products is routinely verified by fluorescent in situ hybridization (FISH). This chromosome-region specific, biotin- labeled genomic DNA is referred to as the target genomic DNA. In the second stage, a cDNA library with unique linker-adapter ends, which we call the source cDNA, is denatured and hybridized in solution with the target genomic DNA which has been denatured and reannealed in the presence of genomic competitor DNA. Under appropriate hybridization conditions, the biotin-labeled target DNA will bind to its homologous counterpart from the source cDNA. The resulting DNA duplexes are captured by streptavidin- coated magnetic beads via the strong affinity between biotin and streptavidin. The cDNAs (referred to as SHACcDNAs) are recovered from their biotin-labeled target genomic counterparts by alkaline denaturation and PCR amplification. The SHAC technique should facilitate the construction of cytogenetically-defined, chromosome region-specific cDNA sublibraries and should assist in the fields of gene discovery and genome mapping. This new technology will be demonstrated using human chromosome bands l3ql2-13 as a test case.
Chromosome region-specific cDNA sublibraries will greatly accelerate molecular genetic research. particularly iii disease gene finding. We plan to develop kits and reagents containing PCR-amplified human chromosome band(s) and tissue- specific cDNA libraries with PCR primer-binding sites separately, so that researchers worldwide can select at will their targeted chromosome region(s) and the cDNA libraries that express this gene(s) of interest.
