Express hybridization of molecular colonies with fluorescent probes

Abstract

DNA colonies formed during PCR in a polyacrylamide gel and RNA colonies grown in an agarose gel containing Qbeta replicase can be identified using the procedure of transfer of molecular colonies onto a nylon membrane followed by membrane hybridization with fluorescent oligonucleotide probes. The suggested improvements significantly simplify and accelerate the procedure. By the example of a chimeric AML1-ETO sequence, a marker of frequently occurring leukemia, the express hybridization method was shown to allow the rapid identification of single molecules and the determination of titers of DNA and RNA targets. Hybridization with a mixture of two oligonucleotide probes labeled with different fluorophores complementary to components of the chimeric molecule ensures the identification of molecular colonies containing both parts of the chimeric sequence and improves the specificity of diagnostics.