An efficient chemical method to generate repetitive sequences depleted DNA probes

Abstract

We describe an efficient method to remove repetitive sequences from DNA of microdissected whole chromosomes, chromosome arms, locus specific probes, and specific bands. The chemical approach described removes human repetitive DNA sequences from a source DNA, eliminating the need for blocking such sequences when the source DNA used as a probe is hybridized with a target DNA. It employs subtracting hybridized biotin-labeled repetitive-sequence DNA complex with phenol and chloroform after incubation of hybridized products with avidin. The method produces unique products that are formed after such repetitive sequences have been removed from the DNA. We have applied our newly designed subtraction strategy to microdissected chromosomes in generating whole chromosome painting (e.g., chromosome 4), chromosome arm (e.g., 1q and 15q), and band (e.g., 3q26) specific probes, and we have demonstrated its utility using flow-sorted chromosome. FISH analyses using these probes show consistent strong signals with no cross-hybridization, and optimal hybridization results can be obtained relatively quickly.