title: 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.
authors.affiliation: Rega Institute for Medical Research, Department of Virology and Chemotherapy, KULeuven-University of Leuven, B-3000 Leuven, Belgium. kaihenrik.dallmeier@rega.kuleuven.be
authors.forename: Kai
authors.initials: K
authors.lastname: Dallmeier
authors.forename: Johan
authors.initials: J
authors.lastname: Neyts
mesh_terms: D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D015005:Yellow fever virus / Q000235:genetics
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger