Method for measuring DNA polymerization and applications of...

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

Reexamination Certificate

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C435S091100, C435S091200, C536S023100, C536S024300, C536S024330

Reexamination Certificate

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07488577

ABSTRACT:
A method for measuring DNA-dependent DNA polymerisation in a biological sample, is described. The method comprises the steps of providing a primer with a single stranded short specific sequence, which is unable to base pair internally, bound to a solid phase; contacting the primer construct with a reaction mixture containing a single stranded deoxynucleotide template with a part of the sequence complementary to the primer and the four deoxynucleoside triphosphates, one of which is modified so that it is specifically recognized by a labeled antibody; adding a biological sample comprising the DNA polymerase, such as retrovirus reverse transcriptase (RT), to the mixture; allowing the polymerase reaction to proceed; incubating the immobilized reaction product with the labeled antibody; detecting the amount of bound labeled antibody; and measuring the amount of incorporated modified deoxynucleoside triphosphate, as a measure of the DNA polymerisation, which may be used drug susceptibility testing. A commercial package is also disclosed.

REFERENCES:
patent: WO 90/06373 (1990-06-01), None
patent: 0101129 (2001-01-01), None
Portsman et al., “A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase,” Journal of Virological Methods, 1991, vol. 31, pp. 181-188.
Rytting et al., “Monoclonal Antibodies to Native HIV Type 1 Reverse Transcriptase and Their Interaction with Enzymes from Different Subtypes,” Aids Research and Human Retroviruses, 2000, vol. 16, No. 13, pp. 1281-1294.

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