Open AccessSimple Method for Selective Amplification of cDNA from a Defined Promoter
Author(s) -
Pavel Boštík,
Lubomír P. Turek,
Thomas H. Haugen
Publication year - 1997
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/97222st02
Subject(s) - complementary dna , microbiology and biotechnology , rapid amplification of cdna ends , biology , ethidium bromide , oligonucleotide , reverse transcriptase , primer dimer , rnase p , rnase h , dna , rna , polymerase chain reaction , gene , genetics , molecular cloning , multiplex polymerase chain reaction
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).