A Guide for In-House Design of Template-Switch-Based 5' Rapid Amplification of cDNA Ends Systems

Anal Biochem. 2010 Feb 15;397(2):227-32. doi: 10.1016/j.ab.2009.10.022. Epub 2009 Nov 1.

Abstract

Rapid amplification of cDNA ends (RACE) is an established strategy used to determine the transcription start point(s) and the 5' untranslated region(s) of mRNA. Different approaches to perform 5' RACE are available, and one particularly simple and powerful strategy is based on a phenomenon called template-switching. We investigated different aspects of template-switch-based 5' RACE, and we describe the different steps leading to the in-house development of a complete 5' RACE system-from oligonucleotide design to polymerase chain reaction (PCR) amplification. We show that the resulting system is reliable, time-efficient, and inexpensive.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Untranslated Regions*
  • DNA, Complementary / genetics*
  • Leukemia Virus, Murine / enzymology
  • Nucleic Acid Amplification Techniques / methods
  • Polymerase Chain Reaction / methods*
  • RNA, Messenger / genetics*
  • RNA-Directed DNA Polymerase / metabolism
  • Reproducibility of Results
  • Templates, Genetic*
  • Transcription Initiation Site

Substances

  • 5' Untranslated Regions
  • DNA, Complementary
  • RNA, Messenger
  • RNA-Directed DNA Polymerase