Rapid Genotyping of 2-bp and 9-bp Deletion Mutations Using the LightCycler Instrument

Abstract

Instead of sequencing the candidate gene PCR fragment, simple methods such as restriction enzyme digestion after PCR are used to detect known mutation. In some cases, however, such methods cannot be applied and sometimes lead to an ambiguous result. A faster method would be required in case of mass screening or emergency. Recently, an elegant method to detect a known mutation with the LightCycler has been introduced. Rapid real-time PCR is monitored by fluorescent oligonucleotide probes that hybridize the target region of the PCR product. Only when the probes hybridize the template fluorescence resonance energy transfer occurs thereby producing a specific fluorescence emission. Then, by slowly heating the PCR product in the probe-hybridized state, the diminishing fluorescence value gives a melting curve. In the presence of mismatch sequences between the template and the probe, the probe melts off at a lower temperature. Therefore, the melting curve clearly demonstrates its genotype as the differences in T_m. This fluorescence PCR on the LightCycler has displayed its marked ability to detect single nucleotide substitution [1, 2]. However, many inherited diseases are caused by small deletion mutations as well as single nucleotide mutations.