REAL TIME DETECTION OF GENETIC SEQUENCES USING A BIPARTITE PROBE
The present invention concerns a method for detection or quantification of a Target Genetic Sequence in a Genetic Sample using a Bipartite Probe. A Bipartite Probe is made of a Target Binding Sequence capable of hybridizing a Target Genetic Sequence and a Nucleic Acid Binding Probe Sequence capable of being transcribed during PCR into the Capture Probe Sequence where it can hybridize with a Signal Generation Molecule. The Signal Generation Molecule participates in the PCR reaction and confers a fluorescent quality to the PCR amplified product. The reaction temperature of each PCR cycle is reduced below the Tm of the Quencher Oligonucleotide thereby allowing quenching of the fluorescence of the non-incorporated Signal Generation Molecule. Fluorescent detection of each PCR cycle establishes a quantitative PCR result.
1 . A method for performing quantitative PCR of a Target Genetic Sequence in a Genetic Sample comprising the steps of:
(1) combining in solution a Bipartite Probe consisting of a target nucleic acid binding sequence capable of hybridizing to a portion of the Target Genetic Sequence with a Nucleic Acid Binding Probe Sequence capable of being transcribed into a Capture Probe Sequence during PCR amplification;
(2) hybridizing a Signal Generation Molecule to the Capture Probe Sequence;
(3) hybridizing a Quencher Oligonucleotide to the non-incorporated Signal Generation Molecule prior to detection at each cycle of PCR; and
(4) generating quantitative PCR Data from each cycle of PCR.
2 . The method of claim 1 wherein said Signal Generation Molecule has its fluorescent signal quenched by said Quencher Oligonucleotide at detection temperature of each PCR cycle.
3 . The method of claim 2 wherein said detection temperature is about or less than the TM of the Quencher Oligonucleotide.
4 . The method of claim 1 wherein said Genetic Sample is genomic DNA.
5 . The method of claim 1 wherein said Genetic Sample is Human.
6 . The method of claim 1 wherein said Genetic Sample is Mouse.
7 . The method of claim 1 wherein said Genetic Sample is Bacterial.
8 . The method of claim 1 wherein more than one quantitative reaction is detected in the same reaction.
9 . The method of claim 7 wherein a Housekeeping allele reaction is performed simultaneously with another reaction.
10 . The method of claim 1 wherein zygosity is determined.
11 . The method of claim 1 wherein SNP zygosity is determined.
12 . The method of claim 1 wherein said Genetic Sample is eukaryotic.
13 . The method of claim 1 wherein said Genetic Sample is prokaryotic.