Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers

Journal Article · Tue Aug 01 04:00:00 EDT 1989 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(89)90420-X· OSTI ID:5026939

Sherman, P A; Fyfe, J A ^[1]

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1. Burroughs Wellcome Company, Research Triangle Park, NC (USA)

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.

• OSTI ID: 5026939
• Journal Information: Analytical Biochemistry; (USA), Journal Name: Analytical Biochemistry; (USA) Vol. 180:2; ISSN ANBCA; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOCHEMICAL REACTION KINETICS
DNA POLYMERASES
ENZYME ACTIVITY
ENZYMES
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
MICROCOCCUS
MICROORGANISMS
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
OLIGONUCLEOTIDES
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOENZYMATIC ASSAY
REACTION KINETICS
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS