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

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.

SUBMITTER: Dallmeier K

PROVIDER: S-EPMC3562438 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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

Dallmeier Kai K Neyts Johan J

Analytical biochemistry 20121030 1

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 depe ...[more]

PMID: 23123427

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