ORIGINAL ARTICLES: Translational researchAn inexpensive, specific and highly sensitive protocol to detect the BrafV600E mutation in melanoma tumor biopsies and bloodPanka, David J.; Sullivan, Ryan J.; Mier, James W.Author Information Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA Correspondence to Dr David J. Panka, PhD, Instructor of Medicine, Harvard Medical School, RW-571, 330 Brookline Avenue, Boston, MA 02215, USA Tel: +1 617 667 0428; fax: +1 617 975 5006 e-mail: [email protected] Received 26 March 2010 Accepted 23 June 2010 Melanoma Research: October 2010 - Volume 20 - Issue 5 - p 401-407 doi: 10.1097/CMR.0b013e32833d8d48 Buy Metrics Abstract The BrafV600E mutation has been detected in patients with metastatic melanoma, colon, thyroid and other cancers. Recent studies suggested that tumors with this mutation are especially sensitive to Braf inhibitors, hence the need to reliably determine the Braf status of tumor specimens. The present technologies used to screen for this mutation fail to address the problems associated with infiltrating stromal and immune cells bearing wild-type Braf alleles and thus may fail to detect the presence of mutant BrafV600E tumors. We have developed a rapid, inexpensive method that reduces the contamination of wild-type Braf sequences from tumor biopsies. The protocol involves a series of PCR amplifications and restriction digestions that take advantage of unique features of both wild type and mutant Braf RNA at position 600. Using this protocol, mutant Braf can be detected in RNA from mixed populations with as few as 0.1% BrafV600E mutant cells. © 2010 Lippincott Williams & Wilkins, Inc.