3' end cDNA amplification using classic RACE.
Having knowledge of the entire 3' sequence of a cDNA is often important because the non-coding terminal region can contain signals that regulate the stability or subcellular localization of the mRNA. Also, some messages use alternative genomic sites for cleavage and polyadenylation that can alter the above properties, or change the encoded protein. Full-length cDNAs can be obtained from complex mixtures of cellular mRNA using rapid amplification of cDNA ends (RACE) PCR as long as part of the mRNA sequence is known; adding non-specific tags to the ends of the cDNA allows the regions between the known parts of the sequence and the ends to be amplified. In 3' RACE, the poly(A) tail functions as a non-specific tag at the 3' end of the mRNA. cDNA ends can be obtained in 1-3 days using this protocol.
Scotto-Lavino
Elizabeth
E
Graduate Program in Molecular & Cellular Pharmacology, Stony Brook University, Stony Brook, New York 11794, USA.
Du
Guangwei
G
Frohman
Michael A
MA
D024509:3' Flanking Region; D018076:DNA, Complementary / Q000138:chemical synthesis*; D011061:Poly A / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012107:Research Design; D048348:Reverse Transcription
D018076:DNA, Complementary; D011061:Poly A