In vitro synthesis of a DNA probe for antisense globin sequences.

Abstract

A DNA probe specific for sequences complementary to globin RNA has been prepared in vitro from globin complementary DNA (cDNA). Globin cDNA was used as the template for the synthesis of a complementary strand (ccDNA) by avian myeloblastosis virus DNA polymerase. After treatment with single strand-specific S1 nuclease, double-stranded globin DNA was denatured in the presence of a vast excess of globin RNA. After hybridization of the cDNA with globin RNA, the ccDNA was isolated by hydroxyapatite chromatography. The ccDNA probe hybridized efficiently to globin cDNA, but not at all to globin RNA. This probe should prove useful in assessing the asymmetry of gene expression in cell-free transcriptional systems.