Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-01-20
2000-07-04
Degen, Nancy
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C12Q 168
Patent
active
060836997
ABSTRACT:
A method is provided for simultaneously determining the positions of a selected nucleotide base in a target region of both strands of a denatured duplex nucleic acid polymer. The nucleic acid polymer is combined with a reactant mixture comprising first and second oligonucleotide primers, said primers binding to the sense and antisense strands, respectively, of the nucleic acid polymer at a location flanking the target region; a thermostable DNA polymerase; a chain-terminating nucleotide triphosphate complementary to the selected nucleotide base; and other reagents for synthesis of chain extension products to form a reaction mixture. This mixture is processed through a plurality of thermal cycles, each including at least a chain extension phase and a denaturation phase to produce chain extension products. These chain extension products are evaluated to determine the positions of the selected bases. The method of the invention differs from the prior art, because the first and second oligonucleotide primers are each labeled with different, spectroscopically-distinguishable fluorescent labels. The method therefore obtains information about both DNA strands simultaneously while providing improved sensitivity as a result of the non-linear increase in the amount of DNA which results from the production of additional templates molecules from unterminated fragments.
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Dunn James M.
Hui May
Lacroix Jean-Michel
Larson Marina T.
Leushner James
Degen Nancy
Larson Thomas G.
Visible Genetics Inc.
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