| US 7,488,577 B2 | ||
| Method for measuring DNA polymerization and applications of the method | ||
| Clas Källander, Uppsala (Sweden); Ingvar Pettersson, Uppsala (Sweden); Simon Gronowitz, Uppsala (Sweden); and Xingwu Shao, Uppsala (Sweden) | ||
| Assigned to Cavidi Tech AB, Uppsala (Sweden) | ||
| Appl. No. 10/479,510 PCT Filed Jun. 14, 2002, PCT No. PCT/SE02/01155 § 371(c)(1), (2), (4) Date Dec. 10, 2003, PCT Pub. No. WO02/103039, PCT Pub. Date Dec. 27, 2002. |
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| Claims priority of provisional application 60/297773, filed on Jun. 14, 2001. | ||
| Prior Publication US 2004/0157230 A1, Aug. 12, 2004 | ||
| Int. Cl. C12Q 1/68 (2006.01); C12P 19/34 (2006.01); C07H 21/02 (2006.01); C07H 21/04 (2006.01) | ||
| U.S. Cl. 435—6 [435/91.1; 435/91.2; 536/23.1; 536/24.3; 536/24.33] | 14 Claims |
| 1. A method for measuring DNA-dependent DNA polymerisation in a biological sample, comprising the steps of
a) providing a primer with a single stranded short specific sequence, which is unable to base pair internally, bound to a
solid phase,
b) contacting the solid phase bound primer with a reaction mixture containing the four deoxynucleoside triphosphates, one
of which is modified so that it is specifically recognized by a labeled antibody, a single stranded deoxynucleotide template
construct composed of, from the 5′ end, a (A)n polymer and a variable part (CTGA)m or (CTG)m, and a sequence complementary
to the primer, and n is greater than zero and m is greater than zero, with the proviso that n may be zero when the template
is (CTGA)m,
c) adding a biological sample comprising the DNA polymerase to the mixture of b),
d) allowing the polymerase reaction to proceed,
e) incubating the immobilized reaction product resulting from d) with the labeled antibody,
f) detecting the amount of bound labeled antibody with the aid of the label used, and
g) measuring the amount of incorporated modified deoxynucleoside triphosphate, as a measure of the DNA polymerization, with
the aid of the label of the bound antibody.
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