Effects of organic solvents on the reverse transcription reaction catalyzed by reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus.
The use of certain organic chemicals has been found to improve yields and specificity in PCR. In this study, we examined the effects of dimethyl sulfoxide (DMSO), formamide, and glycerol on the reverse transcription reaction catalyzed by reverse transcriptases (RTs) from avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV). At 42 °C, DMSO at 24% v/v and formamide at 12-14% inhibited the cDNA synthesis reaction, but DMSO at 12% and formamide at 6-8% improved the efficiency of the cDNA synthesis reaction at low temperatures (25-34 °C). Glycerol at 10% improved the efficiency of the cDNA synthesis reaction at high temperatures (49-61 °C). The effects of DMSO and formamide appeared to be accompanied by decreases in the melting temperatures of the primers, and the effect of glycerol was due to increases in the thermal stabilities of AMV RT and MMLV RT.
Yasukawa
Kiyoshi
K
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan. yasukawa@kais.kyoto-u.ac.jp
Konishi
Atsushi
A
Inouye
Kuniyo
K
D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D002384:Catalysis; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005559:Formamides / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D009930:Organic Chemicals / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000187:drug effects / Q000378:metabolism*; D048348:Reverse Transcription / Q000187:drug effects*; D012997:Solvents / Q000494:pharmacology*
D005559:Formamides; D009930:Organic Chemicals; D012997:Solvents; C031066:formamide; D012194:RNA-Directed DNA Polymerase; D005990:Glycerol; D004121:Dimethyl Sulfoxide