Importance

Loading Importance Indicators...

Abstract

The present invention is directed to a transcription based amplification method for the amplification of DNA targets starting from ds or ssDNA optionally present in a sample, comprising the steps of:--incubating the sample in an amplification buffer with one or more restriction enzymes capable of cleaving DNA at a selected restriction site, said restriction enzyme creating a defined 3' end on the said DNA strand(s), and a promoter-primer, said promoter-primer having a 5' region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3' region complementary to the defined 3' end of the DNA strand, a second primer, having the opposite polarity of the promoter-primer and comprising the 5' end of the target sequence, and in case of ssDNA as the target DNA, a restriction primer;--maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place;--subjecting the sample to a heat treatment at a temperature and time sufficient to inactivate the restricting enzyme and/or to render a double strand single stranded;--adding the following reagents to the sample: an enzyme having RNA dependent DNA polymerase activity, an enzyme having DNA dependent DNA polymerase activity, an enzyme having Rnase H activity, an enzyme having RNA polymerase activity; and--maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for the amplification to take place.

Loading the Abstract Image...

First Claim