Second-strand cDNA synthesis with E. coli DNA polymerase I and RNase H: the fate of information at the mRNA 5' terminus and the effect of E. coli DNA ligase.
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.
D'Alessio
J M
JM
Molecular Biology Research and Development, Life Technologies Inc., Gaithersburg, MD 20877.
Gerard
G F
GF
D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000096:biosynthesis*; D011088:DNA Ligases / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008970:Molecular Weight; D011118:Polynucleotide Ligases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016914:Ribonuclease H
D012333:RNA, Messenger; D004247:DNA; D004256:DNA Polymerase I; D004722:Endoribonucleases; D016914:Ribonuclease H; D011088:DNA Ligases; D011118:Polynucleotide Ligases