A guide for in-house design of template-switch-based 5' rapid amplification of cDNA ends systems.
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.
Pinto
Fernando Lopes
FL
Department of Photochemistry and Molecular Science, Angström Laboratories, Uppsala University, SE-75120 Uppsala, Sweden.
Lindblad
Peter
P
D020121:5' Untranslated Regions; D018076:DNA, Complementary / Q000235:genetics*; D009052:Leukemia Virus, Murine / Q000201:enzymology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D015203:Reproducibility of Results; D013698:Templates, Genetic; D024363:Transcription Initiation Site
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase