Direct synthesis of full length p53 cDNA from frozen liver.
RNA secondary structures can inhibit efficient first strand synthesis in the construction of cDNA clones. Here we report a procedure that incorporates an initial computer analysis of potential secondary structures in related mRNAs. Then a modified, direct reverse transcription-polymerase chain reaction (RT-PCR) procedure optimized for obtaining a full length cDNA starting from a small amount of frozen tissue, without the need for a library construction, is performed. This modified approach is compared to the gene-specific first strand primer method. In addition, the first strand obtained by each of these methods is amplified using two different types of PCR. The utility of this procedure is demonstrated by the successful synthesis of a full length 1.2 kb p53 cDNA by both methods of PCR, from total RNA isolated from woodchuck liver.
Ranganathan
P N
PN
Department of Microbiology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Feitelson
M A
MA
D000818:Animals; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D016158:Genes, p53; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008392:Marmota; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis / Q000737:chemistry; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012313:RNA