Optimization of in vitro transcription and full-length cDNA synthesis using the T4 bacteriophage gene 32 protein.
We evaluated the effect of the T4 bacteriophage gene 32 protein (T4gp32) on in vitro transcription and reverse transcription. T4gp32 doubled the yield of in vitro transcripts obtained with T7 RNA polymerase and increased the yield of cDNA synthesis when used in combination with an RNaseH-deficient Moloney murine leukemia virus [Au: ok] reverse transcriptase. The positive effect could be correlated with the RNA chaperone activity of T4gp32. T4gp32 stimulated the synthesis of long cDNAs, particularly species longer than 7 kb. By comparison, thermal activation of reverse transcriptase with trehalose only boosted the production of shorter cDNAs. For the construction of an Arabidopsis thaliana cDNA library, where the average cDNA size is 1.2 kbp, both the presence of T4gp32 under standard reaction conditions as well as thermal activation resulted in similarly high percentages of full-length cDNA. However, the inclusion of T4gp32 in a standard reverse transcription reaction resulted in the highest cDNA yield. We conclude that the addition of T4gp32 in standard reverse transcription reactions can increase the quality and yield of full-length cDNA libraries.
Piché
Caroline
C
Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Center, 960 Carling Ave. Ottawa, Ontario, K1A0C6, Canada.
Schernthaner
Johann P
JP
D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000502:physiology*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000502:physiology*
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D012194:RNA-Directed DNA Polymerase