Mutant of Moloney murine leukemia virus reverse transcriptase exhibits higher resistance to common RT-qPCR inhibitors.
Inhibitor resistance of several commercial Moloney murine leukemia virus reverse transcriptase (MMLV RT) enzymes was investigated. IC(50) values were determined for potential RNA contaminants, including guanidine thiocyanate, ethanol, formamide, ethylenediaminetetraacetic acid (EDTA), and plant-related acidic polysaccharides. Sensitivity (as judged by MMLV RT IC(50) values) was directly correlated to the outcome of "mock" reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays carried out with exogenous inhibitors. MMLV RT enzymes lacking RNase H activity were shown to be more sensitive to RT-qPCR inhibitors. In contrast, a thermal-resistant MMLV RT pentuple mutant (E69K/E302R/W313F/L435G/N454K) showed higher tolerance to these substances than the wild type. Increased resistance was also noted in RT-qPCR comparisons employing crude cell lysates.
Arezi
Bahram
B
Agilent Technologies, Stratagene Products Division, La Jolla, CA 92037, USA. bahram.arezi@agilent.com
McCarthy
Melissa
M
Hogrefe
Holly
H
D008979:Moloney murine leukemia virus / Q000201:enzymology*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D050505:Mutant Proteins; D018894:Reverse Transcriptase Inhibitors; D012194:RNA-Directed DNA Polymerase