Modified telomeric repeat amplification protocol: a quantitative radioactive assay for telomerase without using electrophoresis.
A polymerase chain reaction (PCR)-based radioactive telomerase assay was developed in our laboratory which is quantitative and does not require electrophoretic evaluation (designated as TP-TRAP; it utilizes two reverse primers). The main steps of the assay include (1) extension of a 20-mer oligonucleotide substrate (MTS) by telomerase, (2) amplification of the telomerase products in the presence of [(3)H]dTTP using the substrate oligonucleotide and two reverse primers (RPC3, 38 mer; RP, 20 mer), (3) isolation of the amplified radioactive dsDNA by precipitation and filtration, (4) determination of the radioactivity of the acid-insoluble DNA. The length of the telomerase products does not increase on amplification. This valuable feature of the assay is achieved by utilization of the two reverse primers and a highly specific PCR protocol. The assay is linear, accurate, and suitable for cell-biological studies where slight quantitative differences in telomerase activity must be detected. The assay is also suitable for screening and characterization of telomerase inhibitors, as shown with a chemically modified oligonucleotide reverse transcriptase inhibitor [(s(4)dU)(35)].
Szatmari
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I
Department of Biochemistry and Molecular Biology, University Medical School of Debrecen, Debrecen, H-4012, Hungary.
Tókés
S
S
Dunn
C B
CB
Bardos
T J
TJ
Aradi
J
J
D002457:Cell Extracts; D002793:Cholic Acids / Q000494:pharmacology; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004983:Ethanolamines / Q000494:pharmacology; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009841:Oligonucleotides / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000032:analysis*; D016615:Telomere / Q000378:metabolism*; D013696:Temperature
D002457:Cell Extracts; D002793:Cholic Acids; D017931:DNA Primers; D003902:Detergents; D004983:Ethanolamines; C414107:FSU oligonucleotide; D009841:Oligonucleotides; D018894:Reverse Transcriptase Inhibitors; D004247:DNA; D019098:Telomerase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C007369:2-diethylaminoethanol