Method for the amplificaton and detection of dna using a transcription based amplification
First Claim
1. Method for the transcription based amplification of a target nucleic acid sequence starting from DNA 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 nucleic acid sequence, 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 restriction 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.
1 Assignment
0 Petitions
Accused Products
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.
-
Citations
7 Claims
-
1. Method for the transcription based amplification of a target nucleic acid sequence starting from DNA 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, andin case of ssDNA as the target nucleic acid sequence, 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 restriction 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. - View Dependent Claims (2, 3, 4, 5, 6, 7)
- end on the said DNA strand(s), and
Specification