SIMPLE METHOD FOR SELECTIVE AMPLIFICATION OF CDNA FROM A DEFINED PROMOTER

A simplified technique for the detection of transcripts from a defined promoter is described. After reverse transcription, a PCR target sequ ence is selectively added to the 3' end of cDNA strands by DNA polymer ase extension directed by an oligonucleotide template. Those cDNA mole cules that do not have ends within a few nucleotides of the promoter s tart site are not extended and thus are excluded from subsequent ampli fication. Even when amplified products are visualized by ethidium brom ide staining of agarose gels, this method requires only 1% of the RNA usually needed for detection of mRNA usually needed for detection of m RNA by standard RNase protection utilizing radiolabeled probes. In con trast to direct detection of cDNA by PCR, this procedure restricts amp lification to a narrow subset of transcripts even when other overlappi ng colinear transcripts are present. We call this detection procedure specific amplification of cDNA ends (SPACE).