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

US6399304B1 · kind B1 · utility

Assignee

• Roche Diagnostics GMBH · DE

Inventors

• Christian Kilger · Hersbruck, DE
• Michael Motz · Heidelberg, DE

Key dates

• Filing date: May 24, 2000
• Grant date: Jun 4, 2002
• Expiry date: May 24, 2020

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q2565/301
• WIPO field: Biotechnology
• WIPO sector: Chemistry

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.