Protocol

Cap-Switching RACE

This protocol was adapted from "Cap-Switching RACE," contributed by Michael A. Frohman, Chapter 25, inPCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

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INTRODUCTION

This method, for the selective amplification of full-length cDNA ends, involves the addition of an adapter during reverse transcription. This method takes advantage of the propensity of Moloney murine leukemia virus reverse transcriptase (MMLV RT) to append two to four cytosines to the 3'-end of newly synthesized cDNA strands. The additional residues are added when the enzyme reaches the 5'-cap structure at the end of the mRNA template (Schmidt and Mueller 1999; Schramm et al. 2000). In the presence of a primer terminating in multiple Gs at its 3'-end, annealing and then complementary copying of the sequence of the annealed . . . [Full Text of this Article]

MATERIALS

Reagents

Equipment

METHOD