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 sequence is selectively added to the 3' end of cDNA strands by DNA polymerase extension directed by an oligonucleotide template. Those cDNA molecules that do not have ends within a few nucleotides of the promoter start site are not extended and thus are excluded from subsequent amplification. Even when amplified products are visualized by ethidium bromide staining of agarose gels, this method requires only 1% of the RNA usually needed for detection of mRNA by standard RNase protection utilizing radiolabeled probes. In contrast to direct detection of cDNA by PCR, this procedure restricts amplification to a narrow subset of transcripts even when other overlapping colinear transcripts are present. We call this detection procedure specific amplification of cDNA ends (SPACE).
Bostik
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P
Department of Veterans Affairs Medical Center, Iowa City, IA, USA.
Turek
L P
LP
Haugen
T H
TH
D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000032:analysis; D018076:DNA, Complementary / Q000032:analysis*; D004996:Ethidium; D006367:HeLa Cells; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D013194:Staining and Labeling; D013698:Templates, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D012333:RNA, Messenger; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D004996:Ethidium