Immunofluorescence experiments were performed as described [67] with minor modifications. Cells were allowed to attach to slides overnight. After fixation with 3.7% paraformaldehyde for 20 min at room temperature, subcellular localization of EYFP fusion proteins was analyzed with a Perkin Elmer spinning disk confocal microscope Ultraview RS system. The microscope used was a Nikon TE2000-U inverted unit with a Nikon 1006 1.4NA DIC lens. The imaging medium was Nikon type A immersion oil. s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor™) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutation-containing DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine. Screening for genetic changes to unveil molecular attributes of human specimens is important for a variety of medical applications, including genotyping for inherited disorders, prediction of the pathologic behavior of malignancies, identification of cancer biomarkers and can affect treatment decisions for individual patients (1) (2) (3) . For example, mutations in genes like EGFR can profoundly influence chemotherapeutic response in lung cancer (2) (3) (4) (5) and the response is modulated by mutations in other genes of the same signaling pathway [e.g. K-ras, HER2, ErbB-3 (1, 6) ]. Therefore there is a need for efficient and high-throughput mutation screening of multiple genes along identified signal transduction pathways in tumor samples. Because a large portion of cancer-causing genetic changes remains unknown and can occur in numerous positions along tumor suppressor genes (e.g. p53, ATM, PTEN) mutation scanning rather than detection of specific mutations is frequently required for molecular cancer profiling.