US 6,399,304 B1 · Sequential activation of one or more enzymatic activities within a thermocycling reaction for synthesizing DNA molecules

Abstract

Method for synthesizing or sequencing a nucleic acid molecule in a thermocycling reaction which initially comprises a nucleic acid molecule, a first primer, a second primer, a reaction buffer, a first thermostable enzyme e.g. a DNA polymerase, (optionally) a thermostable pyrophosphatase, deoxynucleotides or derivatives thereof and in case of a sequencing method a dideoxynucleotide or a derivative thereof and which is characterized in that the thermocycling reaction additionally contains a second thermostable enzyme e.g. a DNA polymerase which, in comparison to the said first thermostable enzyme, exhibits a different enzymatic activity as e.g. has a reduced ability to incorporate dideoxynucleotides as well as the use of the said method. At least one polymerase is initially inhibited whereby the inhibiting agent loses inhibitory ability at cycles of the thermocycling reaction. When both polymerases are inhibited, the inhibiting agents should loose their inhibitory ability at different time points of the reaction.

Claims35

Dependent claims

A method for sequencing nucleic acid in a reaction mixture of a thermocycling reaction wherein the reaction mixture contains at least one nucleic acid molecule and at least one dideoxynucleotide or other terminating nucleotide, said method comprising (1) providing in the thermocycling reaction mixture a first primer (i), a second primer (ii), a reaction buffer, at least one deoxynucleotide or derivative thereof, and at least two thermostable DNA polymerases with different enzyme activities for incorporating dideoxynucleotides;

and (2) activating through elevated temperature at least one of the thermostable DNA polymerases in a later cycle of the thermocycling reaction than another thermostable DNA polymerase.

A kit for sequencing a nucleic acid molecule in a reaction mixture of a thermocycling reaction, said kit comprising

a first primer (i),
a second primer (ii),
a reaction buffer,
at least one deoxynucleotide or derivative thereof,
at least two thermostable DNA polymerases comprising a first thermostable DNA polymerase and a second thermostable DNA polymerase which has a reduced ability to incorporate dideoxynucleotides as compared to the first thermostable DNA polymerase,
wherein the first or second thermostable DNA polymerase is activatable through elevated temperature in a later cycle of the thermocycling reaction compared to the other thermostable DNA polymerase,
at least one dideoxynucleotide or other terminating nucleotide, and
inhibiting agents having a controlled release on the first and second thermostable DNA polymerases, wherein the first and second thermostable DNA polymerases are initially inhibited by the inhibiting agents in the thermocycling sequencing reaction but activatable in later cycles thereof, wherein one of the polymerases is activatable in a later cycle than the other.

5 more elements

Dependencies are inferred from the claim text.

Sequential activation of one or more enzymatic activities within a thermocycling reaction for synthesizing DNA molecules

Life of this patent

Sequential activation of one or more enzymatic activities within a thermocycling reaction for synthesizing DNA molecules

Life of this patent

Abstract

Claims35