Unit

UNIT 4.8 Primer Extension

  1. Steven J. Triezenberg

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb0408s20

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Triezenberg, S. J. 2001. Primer Extension. Current Protocols in Molecular Biology. 20:II:4.8:4.8.1–4.8.5.

Author Information

  1. Michigan State University, East Lansing, Michigan

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: OCT 1992

Abstract

This protocol can be used to map the 5' terminus of an RNA and to quantitate the amount of a given RNA by extending a primer using reverse transcriptase. The primer is an oligonucleotide (or restriction fragment) that is complementary to a portion of the RNA of interest. The primer is end-labeled, hybridized to the RNA, and extended by reverse transcriptase using unlabeled deoxynucleotides to form a single-stranded DNA complementary to the template RNA. The resultant DNA is analyzed on a sequencing gel. The length of the extended primer maps the position of the 5' end of the RNA, and the yield of primer extension product reflects the abundance of the RNA.