DETECTION OF NUCLEIC ACID TARGET SEQUENCES BY ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY

摘要

The present invention relates to methods and kits for detecting the presence of absence of a target DNA sequence, such as a mutation, within an identified region of a selected DNA molecule, such as a gene. In particular aspects, the invention relates to the use of certain aspects of the polymerase chain reaction ("PCR") and ligase chain reaction ("LCR") techniques for the detection of genetic mutations in genes, particularly point mutations. A kit has been developed for direct EPR detection of specific PCR amplified target nucleic acid sequences. The PCR reaction is carried out in the presence of nitroxide-labeled oligomers that are degraded only if hybridized to a complementary target sequence. The degradation of the nitroxide-labeled oligomers into nitroxide-labeled cleavage products results in a characteristic increase of the h-/ho ratio of the EPR signal; in the absence of a complementary target sequence the EPR signal of nitroxide-labeled oligomer remains unchanged.