Shac--Isolation of Chromosome Region Specific CDNA
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AbstractThis proposal describes a method for preparing cDNA sublibraries specific to certain chromosome regprocedure, which we call selection of hybrids by affinity capture (SHAC), can be described in two stbanded mitotic chromosomes are microdissected and PCR- amplified using a universal degenerate primertechnique employed in our lab to obtain chromosome region-specific genomic DNA; the specificity of tis routinely verified by fluorescent in situ hybridization (FISH). This chromosome-region specific,DNA is referred to as the target genomic DNA. In the second stage, a cDNA library with unique linkerwe call the source cDNA, is denatured and hybridized in solution with the target genomic DNA which hand reannealed in the presence of genomic competitor DNA. Under appropriate hybridization conditionstarget DNA will bind to its homologous counterpart from the source cDNA. The resulting DNA duplexesstreptavidin- coated magnetic beads via the strong affinity between biotin and streptavidin. The cDNSHACcDNAs) are recovered from their biotin-labeled target genomic counterparts by alkaline denaturatamplification. The SHAC technique should facilitate the construction of cytogenetically-defined, chrspecific cDNA sublibraries and should assist in the fields of gene discovery and genome mapping. Thibe demonstrated using human chromosome bands l3ql2-13 as a test case.
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