Reaction mixtures for target amplification of nucleic acid with mutant RNA polymerase
View Patent ↗This invention provides a reaction mixture to amplify a nucleic acid. The mixture includes a double stranded DNA molecule with one or more primer recognition sites and a mutant RNA polymerase designed to transcribe deoxynucleotides.
1. A reaction mixture comprising
i. a double stranded DNA comprising a target sequence flanked by a first and second end comprising a phage RNA polymerase recognition sequence on both the first and second ends wherein at least one end has a complementary sequence that forms a phage polymerase recognition site;
ii. a first and second amplification primer each having a phage-encoded RNA polymerase recognition sequence wherein the first primer is complementary to the 5′ end of the target sequence and the second primer is complementary to the antisense sequence of the 3′ end of the target sequence;
iii. phage-encoded RNA polymerase mutated to recognize and polymerize dNTP; and,
iv. an excess of dNTP.
2. A reaction mixture of claim 1 wherein both ends of the DNA have phage polymerase recognition sites.
3. A reaction mixture of claim 1 wherein both ends of the DNA have identical phage polymerase recognition sites.
4. A reaction mixture of claim 1 wherein the DNA comprises a signature sequence for a specific genus or species of organism.
5. A reaction mixture of claim 1 wherein one end of the DNA has a complementary sequence that forms a phage polymerase recognition site.