3' RACE walking along a large cDNA employing tiered suppression PCR.
Large genes present particular cloning difficulties, especially when expressed at relatively low levels. We describe a novel method, termed 3' rapid amplification of cDNA ends (RACE) walking, for the rapid determination of unknown 3' flanking sequence of a large cDNA. The technique is a derivative of the anchored PCR 5' RACE procedure but includes a specific and limited second-strand cDNA synthesis and a tiered "panhandle" suppression of nonspecific products. The method generated 900 bp of new sequence for the large tammar wallaby ATRY gene in two easy steps, in which standard 3' RACE and PCR-based cDNA library walking proved unsuccessful. This robust approach represents a new tool for isolating unknown sequence under challenging cloning scenarios such as poor library representation, long coding regions, long 3' untranslated regions, and difficult template regions.
Park
Daniel J
DJ
Gate 12, Royal Parade, Dept. Zoology, Melbourne University, VIC 3010, Australia. d.park@unimelb.edu.au
Pask
Andrew J
AJ
Renfree
Marilyn B
MB
Graves
Jennifer A Marshall
JA
D000818:Animals; D001483:Base Sequence; D016386:Chromosome Walking / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry* / Q000235:genetics; D007614:Macropodidae / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D018076:DNA, Complementary