Oligonucleotide fingerprinting with (CAC)5: nonradioactive in-gel hybridization and isolation of individual hypervariable loci

Abstract

The first topic to be treated in this paper is the nonradioactive DNA fingerprinting by means of in-gel hybridization with digoxigenated (CAC)5. Besides the fact that time-consuming Southern blotting can be avoided, the dried agarose is an excellent matrix to produce background-free nonradioactive DNA fingerprints. There is no tendency of either the oligonucleotide probe or the antibody towards unspecific binding to the dried agarose. Prehybridization and blocking steps are therefore superfluous. Furthermore, we will discuss what effect the degree of crosslinking of the antibody-enzyme conjugates has. The second topic concerns the isolation and characterization of locus-specific probes from a human (CAC)5 fingerprint. The isolation and characterization of one variable probe, by screening complete genomic libraries, is described and discussed. This probe is compared to a hypervariable single-copy probe, isolated from a size-enriched genomic library. The sequence of the repeat flanking locus-specific probe is presented and a semi-specific, adaptor-mediated polymerase chain reaction was designed to amplify (CAC)n/(GTG)n flanking sequences.