METHODS AND COMPOSITIONS FOR TRANSCRIPTION-BASED NUCLEIC ACID AMPLIFICATION
First Claim
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.
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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.
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Citations
22 Claims
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17)
- DNA portion;
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12. A kit comprising one or more containers comprising a first primer comprising an RNA/DNA composite primer, 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.
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18. A system for amplifying a sequence of interest comprising:
- (a) a first primer which is a composite primer, (b) a second primer, (c) a DNA dependent DNA polymerase, (d) and RNA dependent DNA polymerase, (e) a propromoter polynucleotide, (f) an RNA polymerase, and (g) an enzyme which cleaves RNA from an RNA/DNA hybrid.
- View Dependent Claims (19, 20, 21, 22)
Specification