Method for the direct, exponential amplification and sequencing of DNA molecules and its application

US6605428B2 · kind B2 · utility

Assignee

• Roche Diagnostics GMBH · DE

Inventors

• Christian Kilger · Hersbruck, DE
• Svante Paabo · Hersbruck, DE

Key dates

• Filing date: Jun 24, 1999
• Grant date: Aug 12, 2003
• Expiry date: Jun 24, 2019

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/6869
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A method is described for the direct, exponential amplification and sequencing (“DEXAS”) of a DNA molecule from a complex mixture of nucleic acids, wherein truncated DNA molecules as well as DNA molecules of full length are synthesized simultaneously and exponentially between two positions on the said DNA molecule, which initially contains a DNA molecule in a thermocycling reaction, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, a thermostable pyrophosphatase (optionally), deoxynucleotides or derivatives thereof and a dideoxynucleotide or derivatives thereof. In a preferred embodiment of the method of the invention, direct sequencing of RNA can be performed using one polymerase having a Tabor-Richardson mutation, or a functional derivative thereof, and reverse transcriptase activity. In a more preferred embodiment of the method of the invention, direct sequencing of RNA can be performed in one step, in one vessel.