Express Hybridization of Molecular Colonies with Fluorescent Probes

E. V. Chetverina^1 #, A. V. Kravchenko¹, M. V. Falaleeva¹ and A. B. Chetverin¹

^#Phone: (4967) 73-89-17; fax: (495) 632-78-71; e-mail: helena@vega.protres.ru

¹Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290, Russia

Received: June 30, 2006;  in final form: October 3, 2006

Abstract.  DNA colonies formed during PCR in a polyacrylamide gel and RNA colonies grown in an agarose gel containing Qβ 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 shorten 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.

Key words:  chimeric RNA markers, fluorescent probes, membrane hybridization, leukemia, molecular colony technique, molecular diagnostics

Russian Journal of Bioorganic Chemistry 2007, 33 (4): 423-430