Patent ID: 6063603
Filing Date: 2000-05-16
Classification: C12Q

Abstract:
A kit for the isothermal amplification of a specific nucleic acid sequence or its complement according to a process for the amplification of an RNA first template or a second DNA template as defined herein, at a relatively constant temperature and without serial addition of reagents, said process comprising(A) providing a single reaction medium including a first primer, a second primer having an anti-sense sequence of a promoter, an RNA polymerase that recognizes said promoter, and a DNA polymerase, wherein the ribonuclease activity of said DNA polymerase hydrolyzes the RNA of an RNA-DNA hybrid without hydrolyzing single or double-stranded RNA or DNA;(B) providing in said single reaction medium said RNA first template, such that the following steps take place:(1) hybridizing said first primer to said RNA first template,(2) synthesizing from said DNA polymerase and said RNA first template a DNA second template by extending said first primer and thereby forming an RNA-DNA hybrid intermediate,(3) hydrolyzing the RNA of said RNA-DNA hybrid intermediate,(4) hybridizing said second primer to said DNA second template,(5) synthesizing from said DNA polymerase, said second primer, and said DNA second template a functional promoter recognized by an RNA polymerase,(6) having said RNA polymerase recognizing said functional promoter and transcribing said DNA second template, thereby providing copies of said RNA first template,and thereafter(C) maintaining said conditions for a time sufficient to achieve a desired amplification of said RNA first template or said DNA second template, said kit comprising one or more receptacles containing(i) a first primer,(ii) a second primer comprising an anti-sense sequence of a promoter recognized by an RNA polymerase,(iii) a RNA polymerase that recognizes said promoter,(iv) a DNA polymerase, wherein the ribonuclease activity of said DNA polymerase hydrolyzes the RNA of an RNA-DNA hybrid without hydrolyzing single or double-stranded RNA or DNA, and(v) a ribonuclease, with ribonuclease activity that is separate and distinct from the ribonuclease activity of said DNA polymerase wherein said ribonuclease hydrolyzes the RNA of an RNA-DNA hybrid without hydrolyzing single or double-stranded RNA or DNA.