Open AccessSecond-strand cDNA synthesis withE.coliDNA polymerase I and RNase H: the fate of information at the mRNA 5' terminus and the effect ofE.coliDNA ligase
Author(s) -
James M. D’Alessio,
Gary F. Gerard
Publication year - 1988
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/16.5.1999
Subject(s) - biology , complementary dna , microbiology and biotechnology , primer (cosmetics) , rnase h , dna polymerase i , rnase p , rna , oligonucleotide , rapid amplification of cdna ends , cloning (programming) , dna , molecular cloning , genetics , reverse transcriptase , gene , chemistry , organic chemistry , computer science , programming language
A simple method for generating cDNA libraries has been described (1) in which RNase H-DNA polymerase I-mediated second-strand cDNA synthesis primes from an RNA oligonucleotide derived from the 5' (capped) end of mRNA. The size of this oligonucleotide and the fate of the information corresponding to the RNA during subsequent cloning have not been established. We show here that the 5'-most RNA primer varies in length from 8 to 21 nucleotides, and that information corresponding to the length of the RNA primer is normally lost during cloning. A modification of the second-strand cDNA synthesis procedure is described which allows cloning of all, or almost all, of the primer sequence information. In addition, we show that the presence of E. coli DNA ligase during second-strand cDNA synthesis can increase the length of the cDNA clones obtained from long RNAs. Cloning by addition of linkers provides the greatest chance of obtaining near full-length cDNA clones from long mRNAs.