Detection of nucleic acid target sequences by electron paramagnetic resonance spectroscopy

A method for detecting the presence or absence of a target nucleic acid sequence in a sample, said method comprising:

• a) providing in a PCR reaction mixture a sample suspected to contain a target nucleic acid sequence, oligonucleotide PCR primers that hybridize to the target nucleic acid sequence for PCR amplification of said target sequence, each of four deoxynucleoside triphosphates, nucleic acid polymerase having 5′ to 3′ exonuclease activity and devoid of 3′ to 5′ exonuclease activity, and an EPR-labeled oligonucleotide analytical probe (EPR probe);
• b) amplifying target nucleic acid sequence in the sample under suitable PCR reaction mixture temperature conditions by a repetitive series of PCR thermal cycling steps comprising:
• 1) denaturing the target nucleic acid sequence into opposite strands;
• 2) hybridizing the EPR probe within the target nucleic acid sequence of the denatured strands and hybridizing the oligonucleotide PCR primers to the denatured strands, and
• 3) extending the hybridized primers with the four deoxynucleoside triphosphates and the nucleic acid polymerase, and producing 5′ EPR labeled nucleotide fragments during this extension phase by the 5′ to 3′ exonuclease activity of the nucleic acid polymerase on the EPR probes annealed to the denatured strands;
• and
• c) measuring the EPR resonance signals produced by degradation of EPR-labeled probes by magnetic resonance as a measure of the presence of the target nucleic acid sequence following amplification of the target nucleic acid sequence by one or more series of thermal cycling steps;
• wherein the 5′ EPR labeled nucleotide fragments produced are measured by measuring EPR signal h0 and h−1 peaks and detecting a change in the original h−1/h0 ratio.