Advertisement

Determination of Transcription Start Sites (TSSs) in Yersinia pestis with a Primer Extension Assay

  • Yanping Han
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

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 words

Yersinia pestis Transcription start site Autoradiography Primer extension 

References

  1. 1.
    Carey MF, Peterson CL, Smale ST (2013) The primer extension assay. Cold Spring Harb Protoc 2013(2):164–173CrossRefPubMedGoogle Scholar
  2. 2.
    Dalmasso A, Civera T, Bottero MT (2009) Multiplex primer-extension assay for identification of six pathogenic vibrios. Int J Food Microbiol 129(1):21–25CrossRefPubMedGoogle Scholar
  3. 3.
    Storz G, Vogel J, Wassarman KM (2011) Regulation by small RNAs in bacteria: expanding frontiers. Mol Cell 43(6):880–891CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Gottesman S, Storz G (2011) Bacterial small RNA regulators: versatile roles and rapidly evolving variations. Cold Spring Harb Perspect Biol 3(12).  https://doi.org/10.1101/cshperspect.a003798 Google Scholar
  5. 5.
    Zhang Y, Wang L, Han Y, Yan Y, Tan Y, Zhou L, Cui Y, Du Z, Wang X, Bi Y et al (2013) Autoregulation of PhoP/PhoQ and positive regulation of the cyclic AMP receptor protein-cyclic AMP complex by PhoP in Yersinia pestis. J Bacteriol 195(5):1022–1030CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Li Z, Lan X, Han R, Wang J, Huang Y, Sun J, Guo W, Chen H (2017) miR-2478 inhibits TGFbeta1 expression by targeting the transcriptional activation region downstream of the TGFbeta1 promoter in dairy goats. Sci Rep 7:42627CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Yanping Han
    • 1
  1. 1.Beijing Institute of Microbiology and EpidemiologyBeijingChina

Personalised recommendations