Methods And Compositions For Transcription-Based Nucleic Acid Amplification
Methods for isothermal exponential amplification of a target polynucleotide are disclosed. The methods employ two transcription modules, the first module providing linear amplification resulting in RNA transcripts, and a second module providing for further (generally cyclical) amplification resulting in more RNA transcripts. In one aspect, the amplification of the first module is composite primer based. In a second aspect, the amplification of the first module is based on target switching to generate a primer extension product comprising a promoter sequence. In all aspects, the RNA transcripts of the first transcription module are subjected to further amplification by creating an intermediate product comprising a double stranded promoter region from which transcription can occur. The invention further provides compositions and kits for practicing said methods, as well as methods which use the amplification results.
1 . A kit comprising (a) a propromoter template switch oligonucleotide (TSO), (b) a first primer, (c) a DNA dependent DNA polymerase, (d) an RNA dependent DNA polymerase, (e) an enzyme that cleaves RNA from an RNA/DNA hybrid, and (f) an RNA polymerase.
2 . The kit of claim 1 further comprising a second primer.
3 . The kit of claim 1 wherein the enzyme which cleaves RNA from an RNA/DNA hybrid has RNAse H activity.
4 . A kit comprising one or more containers comprising a first primer comprising a propromoter TSO, a first primer, an enzyme which cleaves RNA from an RNA/DNA hybrid and any two of: (a) a second primer, (b) an enzyme with DNA dependent DNA polymerase activity, (c) an enzyme with RNA dependent DNA polymerase activity, and (d) an RNA polymerase.
5 . The kit of claims 1 or 4 further comprising deoxynucleoside triphosphates or ribonucleoside triphosphates.
6 . The kit of claim 5 wherein a deoxynucleoside triphosphate or a ribonucleoside triphosphate is labeled.
7 . The kit of claim 1 or 4 further comprising a nucleotide analog that upon incorporation into a primer extension product effects termination of nucleotide polymerization.
8 . The kit of claim 7 wherein the nucleotide analog which effects termination is labeled.
9 . The kit of claims 1 or 4 wherein one or more components is provided as a dry powder.
10 . A system for amplifying a sequence of interest, comprising: (a) a propromoter TSO; (b) a first primer; (c) an RNA-dependent DNA polymerase; (d) an enzyme that cleaves RNA from an RNA/DNA hybrid; and (e) an RNA polymerase.
11 . The system of claim 10 further comprising a DNA-dependent DNA polymerase.
12 . The system of claim 10 , wherein the RNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.
13 . The system of claim 11 , wherein the DNA-dependent DNA polymerase and the RNA-dependent DNA polymerase are the same enzyme.
14 . The system of claim 11 , wherein the DNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.
15 . The system of claim 11 , wherein the RNA-dependent DNA polymerase, DNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same.
16 . The system of claim 10 further comprising a second primer.
17 . The system of claim 10 wherein the first primer comprised DNA.