Amplification of midivariant dna templates

Details Amplification of midivariant dna templates Amplification of midivariant dna templates

C - Chemistry, Metallurgy

12

N

C12N 15/10 (2006.01) C07H 21/04 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2046713

ABSTRACT New methods are provided for the amplification of a midivariant DNA containing an inserted target specific nucleic acid sequence(s) to enable detection of the presence of a target nucleic acid sequence in a test sample. One method employs midivariant DNA as a template for the synthesis of RNA catalyzed by QB replicase. Two midivariant DNA/probe conjugates including a nonreplicable portion of midivariant DNA and a target specific nucleic acid sequence (probe) are described. An amplification method including the steps of hybridization and ligation of the midivariant DNA/probe conjugates followed by replication of the DNA template has enabled detection of less than 200 template molecules. In a modified amplification method one of the midivariant DNA/probe conjugates further includes a RNA polymerase promoter sequence to enable transcription of the midivariant DNA template into RNA. The sequential ligation-transcription-replication method enables the detection of a single template molecule. The detectable molecule(s) are indicative of the presence of the target nucleic acid sequences in the test sample.

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