High-sensitivity S1 mapping with single-stranded [32P]DNA probes synthesized from bacteriophage M13mp templates

Abstract

A method is described by which high-specific-activity single-stranded (ss) [alpha-32P]DNA of a defined size complementary to sequences cloned into bacteriophage M13 is synthesized. The ss DNA template is annealed with a universal sequencing primer, the primer extended with DNA polymerase I Klenow fragment and the DNA duplex cut at a unique site 5' to the multiple cloning sites in the M13 phage. The reaction products are denatured and the ss alpha-32P probe fragment complementary to the cloned sequence is separated from the template by electrophoresis. The utility of such probes for S1 mapping is shown by mapping the 3' ends of transcripts from a mutant Drosophila heat-shock gene. The method described here is up to 300 times more sensitive than conventional S1 mapping techniques.