Nucleic Acids Research Advance Access published online on June 1, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm403
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Genomics |
s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis
1Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA, 2Physics and Department of Medical Oncology, Department of Radiation Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA, 3Translational Research Laboratory: Center for Clinical and Translational Research, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA and 4Lowe Center for Thoracic Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA
*To whom correspondence should be addressed. Tel: +1-617-525-7122; Fax: +1-617-587-6037; Email: mmakrigiorgos{at}partners.org
Received March 1, 2007. Accepted May 2, 2007.
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 (SurveyorTM) 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.
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