Bioluminometric method for real-time detection of reverse transcriptase activity.
A simple and sensitive technique for detection of reverse transcriptase (RT) activity in real time has been developed. The technique is based on continuous detection of the inorganic pyrophosphate formed in the RT-catalyzed reaction by a luminometric method. The technique has been used for continuous monitoring of RT-catalyzed DNA synthesis on both homo- and heteropolymeric templates. The assay is sensitive and yields linear responses between 1.5-960 mU of avian myeloblastosis virus RT (AMV-RT). The assay was used for detection of the inhibitory effect of dideoxythymidine (ddTTP) on the AMV-RT activity and also for real-time detection of single-base incorporation events catalyzed by AMV-RT. The possibility of using the new technique for other applications is discussed.
Karamohamed
S
S
Royal Institute of Technology, Stockholm, Sweden.
Ronaghi
M
M
Nyrén
P
P
D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D004247:DNA / Q000096:biosynthesis; D003854:Deoxyribonucleotides / Q000378:metabolism; D054306:Dideoxynucleotides; D011756:Diphosphates / Q000032:analysis; D007700:Kinetics; D008163:Luminescent Measurements; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000494:pharmacology
D003854:Deoxyribonucleotides; D054306:Dideoxynucleotides; D011756:Diphosphates; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D004247:DNA; D012194:RNA-Directed DNA Polymerase; C017216:2',3'-dideoxythymidine triphosphate