Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers.
The enzymatic assay for deoxyribonucleoside triphosphates has been improved by using synthetic oligonucleotides of a carefully defined sequence as template primers for DNA polymerase. High backgrounds, which limit the sensitivity of the assay when calf thymus DNA or alternating copolymers are used as template primers, were eliminated with these oligonucleotide template primers. Sensitivity was further increased by designing the template primer to incorporate multiple labeled deoxyribonucleotides per limiting unlabeled deoxyribonucleotide. Each of several DNA polymerases exhibited unique reaction characteristics with the oligonucleotide template primers, which was attributed to the differing exonuclease activities associated with these various enzymes. Assay optimization therefore included matching the polymerase with the template primer to obtain the lowest background reaction and highest sensitivity. This modified assay is particularly well suited for keeping cell sample size to a minimum in experimental protocols which generate large numbers of data points or require careful timing of sampling. With this technique, we measured the levels of all four deoxyribonucleoside triphosphates in extracts from as few as 2 x 10(4) cultured cells.
Sherman
P A
PA
Experimental Therapy Division, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709.
Fyfe
J A
JA
D001483:Base Sequence; D002478:Cells, Cultured; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000032:analysis; D003845:Deoxycytosine Nucleotides / Q000032:analysis; D003848:Deoxyguanine Nucleotides / Q000032:analysis; D003854:Deoxyribonucleotides / Q000032:analysis*; D006801:Humans; D008837:Micrococcus / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics*; D010472:Perchlorates / Q000494:pharmacology; D011067:Poly dA-dT / Q000378:metabolism; D012015:Reference Standards; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000032:analysis; D014316:Tritium
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D009841:Oligonucleotides; D010472:Perchlorates; D013942:Thymine Nucleotides; D014316:Tritium; C024107:2'-deoxycytidine 5'-triphosphate; D011067:Poly dA-dT; C029603:deoxyguanosine triphosphate; D004259:DNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate