Methods and compositions for transcription-based nucleic acid amplification
View Patent ↗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 first composite primer for hybridization to a target polynucleotide, wherein said composite primer comprises an RNA portion and a 3′ DNA portion; a propromoter polynucleotide; and a second primer.
2. The kit of claim 1 further comprising a polynucleotide comprising a termination polynucleotide sequence.
3. The kit of claim 1 wherein the polynucleotide comprising a termination polynucleotide sequence is a template switch oligonucleotide (TSO).
4. The kit of claim 1 further comprising an enzyme which cleaves RNA from an RNA/DNA hybrid.
5. The kit of claim 4 wherein the enzyme which cleaves RNA from an RNA/DNA hybrid has RNAse H activity.
6. The kit of claim 1 further comprising an enzyme with DNA dependent DNA polymerase activity.
7. The kit of claim 1 further comprising an enzyme with RNA dependent DNA polymerase activity.
8. The kit of claim 1 further comprising one or more enzymes with all of DNA dependent DNA polymerase activity, RNA dependent DNA polymerase activity, and RNAse H activity.
9. The kit of claim 1 further comprising an RNA polymerase.
10. The kit of claim 1 wherein, in the composite primer, the RNA portion is adjacent to the DNA portion.
11. The kit of claim 1 wherein the composite primer comprises 5′ and 3′ DNA portions with at least one intervening RNA portion.
12. A kit comprising one or more containers comprising a first primer comprising an RNA/DNA composite primer, wherein said composite primer comprises an RNA portion and a 3′ DNA portion; a propromoter polynucleotide 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 (d) an RNA polymerase, and (e) an enzyme which cleaves RNA from an RNA/DNA hybrid.
13. The kit of claim 1 or 12 further comprising deoxynucleoside triphosphates or ribonucleoside triphosphates.
14. The kit of claim 13 wherein a nucleoside triphosphate or a ribonucleoside triphosphate is labeled.
15. The kit of claim 1 or 12 further comprising a nucleotide analog that upon incorporation into a primer extension product effects termination of nucleotide polymerization.
16. The kit of claim 15 wherein the nucleotide analog which effects termination is labeled.
17. The kit of claim 1 or 12 wherein one or more components is provided as a dry powder.
18. A system for amplifying a sequence of interest comprising: (a) a first primer which is a composite primer, wherein said composite primer comprises an RNA portion and a 3′ DNA portion, (b) a second primer, (c) a DNA dependent DNA polymerase, (d) an RNA dependent DNA polymerase, (e) a propromoter polynucleotide, (f) an RNA polymerase, and (g) an enzyme which cleaves RNA from an RNA/DNA hybrid.
19. The system of claim 18 further comprising a polynucleotide comprising a termination polynucleotide sequence.
20. The system of claim 18 wherein the DNA-dependent DNA polymerase and the RNA-dependent DNA polymerase are the same enzyme.
21. The system of claim 18 wherein the DNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.
22. The system of claim 18 wherein the RNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.