Simple and inexpensive three-step rapid amplification of cDNA 5′ ends using 5′ phosphorylated primers

Kai Dallmeier,Johan Neyts

doi:10.1016/j.ab.2012.10.031

Abstract

Rapid amplification of cDNA 5′ ends (5′-RACE) is routinely used for the sequence analysis of the upstream noncoding regions of cellular mRNAs; however, it represents a tedious and cost-intensive procedure. By employing 5′ phosphorylated gene-specific primers for first-strand cDNA synthesis, we cut short the previously established reverse ligation and amplification protocol of Mandl and coworkers (BioTechniques, 1991, vol. 10, pp. 484–486) to a streamlined three-step procedure that no longer depends on enzymatic mRNA decapping or linker ligation. The novel three-step protocol has been validated by mapping the transcriptional start sites of heterologously expressed yellow fever virus genomic RNAs from cultured mammalian cells.

Highlights

Rapid amplification of cDNA 50 ends (50-Rapid amplification of cDNA ends (RACE)) for an eukaryotic mRNA is a multistep procedure that includes (i) removal of the 50 7-methyl-guanosine (7mGppp) cap by tobacco acid pyrophosphatase (TAP), (ii) ligation of an oligoribonucleotide linker to the resulting 50-phosphate by the activity of the phage T4 RNA ligase 1 (RNL1), (iii) reverse transcription by an RNA-dependent DNA polymerase that uses as a primer an oligodesoxynucleotide (ODN) complementary to the synthetic RNA linker added in step (ii) and (iv) subsequent polymerase chain reaction (PCR) on this first-strand cDNA with the linker-specific primer already used in step (iii) and a second gene-specific reverse primer
A method introduced by Mandl and coworkers [6] for the simultaneous cloning of the RNA 50 and 30 ends needs to be considered as a variant of this general four-step protocol, but substituting ligation of a synthetic RNA linker by an intramolecular ligation (RNA circularization) prior to subsequent RT–PCR using a pair of inverted gene-specific ODN primers (Fig. 1A)
The novel streamlined three-step 50-RACE employs the following steps: (i) reverse transcription with 50 phosphorylated genespecific primers, (ii) cDNA circularization by RNL1 and (iii) PCR amplification using inverted primers followed by blunt end cloning for sequencing

Summary

Introduction

The novel three-step protocol has been validated by mapping the transcriptional start sites of heterologously expressed yellow fever virus genomic RNAs from cultured mammalian cells.

Results

Conclusion