The investigators propose to further develop high-resolution fluorescent melting curve analysis for nucleic acids. Melting curve analysis is most conveniently performed immediately after amplification by PCR. Products can be identified by characteristic melting profiles that can distinguish even single base changes. The LightCycler (TM) is the applicants' commercial platform for thermal cycling and fluorescent monitoring. The investigators will alter the LightCycler to achieve slow (0.01degrees /sec) and steady (+/- 0.01degrees C) temperature transitions by modifying hardware, temperature sensing, and control mechanisms. Such gradual transitions approach equilibrium conditions and allow high-resolution melting curves, similar to those obtained by absorbency measurements. The increased resolution obtained with slow melting will be demonstrated by hybridization probes and fluorescence resonance energy transfer in model systems. The degree of multiplexing by probe/allele melting temperatures depends on the melting curve resolution and temperature stability. The effect of mismatch position and probe length on single base mismatch discrimination from a perfectly matched probe will be studied to assess the potential of this method for mutation scanning. Finally, high-resolution melting curves of long DNA will be obtained with the double-stranded DNA dye SYBR Green I and the melting domains compared to absorbency techniques.
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| Erali, Maria; Wittwer, Carl T (2010) High resolution melting analysis for gene scanning. Methods 50:250-61 |
