Quantitative determination of a DNA polymerase and a test kit us

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

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536 27, 935 77, 935 78, C12Q 168, C07H 1512, C12N 1500

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active

050494902

ABSTRACT:
A convenient method for the quantitative determination of a DNA polymerase includes contacting an aqueous test specimen suspected of containing the enzyme with the following: a single-stranded DNA template present in a concentration of at least about 10.sup.-8 molar bases, a DNA primer complementary to the template, a source of a metal polymerase cofactor, sufficient deoxyribonucleoside triphosphates to synthesize double-stranded DNA in the presence of the polymerase, and a colorimetric or fluorescent dye which is capable of providing a detectable signal when a primed single-stranded nucleic acid is converted to double-stranded DNA by the polymerase. The rate of signal generation is then measured and can be correlated with the level of DNA polymerase in the specimen using graphical or mathematical means. The results of this method are precise, having a covariance of less than about 10%. A test kit includes the reagents needed for carrying out this method.

REFERENCES:
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patent: 4683202 (1987-07-01), Mullis
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"Amplifications", Perkin Elmer Cetus Bulletin, pp. 8-10 (Feb. 1989).
West et al., Anal. Biochem., 147, pp. 289-295 (1985).
Cesavone et al., Anal. Biochem., 100, pp. 188-197 (1979).
Stout et al., Anal. Biochem., 127, pp. 302-307 (1982).

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