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Direct synthesis of full length p53 cDNA from frozen liver
Author(s) -
Ranganathan Perungavur N.,
Feitelson Mark A.
Publication year - 1997
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216549700201901
Subject(s) - complementary dna , primer (cosmetics) , microbiology and biotechnology , rapid amplification of cdna ends , cdna library , reverse transcriptase , biology , polymerase chain reaction , rna , reverse transcription polymerase chain reaction , gene , chemistry , messenger rna , genetics , molecular cloning , organic chemistry
Abstract 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.