Methods And Compositions For Transcription-Based Nucleic Acid Amplification

Abstract

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.