Reverse transcription of heterologous RNA by RNA-dependent DNA polymerase from Escherichia coli
Vorob'eva, N.V.; Nebrat, L.T.; Potapov, V.A.; Romashchenko, A.G.; Salganik, R.I.
Molekuliarnaia Biologiia 16(5): 977-983
1982
ISSN/ISBN: 0026-8984 PMID: 6183576 Document Number: 185659
The appropriate conditions for the reverse transcription of rabbit globin mRNA by E. coli RNA-dependent DNA polymerase has been studied. By reducing the ionic strength, increasing the incubation temperature from 37 to 43 degrees and predenaturing the template it was possible to increase the cDNA size. The molar ratio of Mn2+ and dNTP optimal for cDNA synthesis is approximately 1.5-1.7:1. The increase of dNTP concentration from 0.05 to 0.4 mM each, under conditions of Mn2+ deficiency, results in the decrease of the cDNA size to 4S, obviously, the inhibitory effect of dNTP is not complexed with Mn2+. The synthesis of cDNA is inhibited also by the excess of Mn2+. Hybridization of cDNA with globin mRNA protects the former from S1-nuclease. Optimization of the conditions for reverse transcription of heterologous RNa by E. coli RNA-dependent DNA polymerase led to the increase of the cDNA length up to approximately 550 nucleotides which is about 70% of the RNA template length.