UNIT 4.8 Primer Extension
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Triezenberg, S. J. 2001. Primer Extension. Current Protocols in Molecular Biology. 20:4.8.1–4.8.5.
- Published Online: 1 MAY 2001
- Published Print: OCT 1992
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.