Determination of Transcription Start Sites (TSSs) in Yersinia pestis with a Primer Extension Assay
The transcription start site of a gene can be experimentally determined by identifying the 5′ end of the RNA transcript in a primer extension assay. The protocol begins with a reverse primer, which is annealed to the specific mRNA molecule and is 5′ end labeled with [γ-32P] ATP. The cDNA product is obtained by elongating the primer attached to the 5′ end of the mRNA. Sequencing reactions produces a series of adjacent PCR products, which provide a measure of the distance from the 5′ end of the synthetic oligonucleotide to the beginning of the mRNA transcript, and is compared with the cDNA product described above. The cDNA products are analyzed with denaturing polyacrylamide gel electrophoresis, followed by autoradiography. The film is read to determine the 3′ end of the cDNA, which is complementary to the 5′ end of the mRNA.
Key wordsYersinia pestis Transcription start site Autoradiography Primer extension