Melting Curve Assay Design. PCR primers are designed within introns of the functional KRAS (Ki-Ras2) gene on chromosome 12 to avoid amplification of a KRAS (Ki-Ras1) pseudogene on chromosome 6. A Cy5.5-labeled wild type sensor probe sits over the mutation hotspot at codons 12 and 13. When a KRAS mutation is present, the sensor probe melts off at a lower Tm.
Pritchard et al. BMC Clinical Pathology 2010 10:6 doi:10.1186/1472-6890-10-6