Mutation Detection Service Details
Project and Assay Design Workflow
We will first have a meeting to discuss your research goals and the suitability of traditional PCR and Sanger sequencing technology in achieving those goals. If this platform is applicable, we will use a Primer 3-based software to design PCR primers for your region of interest, using the most current build in dbSNP. These primers will be tailed with M13 seqeunces in order to facilitate the downstream Sanger sequencing process. We will then optimize thermal cycling conditions using our internal human or mouse controls. If your lab has samples of known mutation status, we may ask for aliquots of those samples during the validation stage.
Sample Processing Workflow
Once samples are received, LMT staff will determine the concentration via NanoDrop. An aliquot of the sample will be normalized and used as template for PCR or RT-PCR. Resulting amplicons are analyzed for size and concentration using Lab-on-a-Chip technology. Products are purified via enzymes (Exonuclease and Shrimp Alkaline Phosphatase) and cycle sequenced. Once purified by magnetic beads (Agencourt’s CleanSeq®), the reactions are loaded onto the ABI 3730xl sequencing instrument. Following electrophoresis, chromatogram data will be analyzed using our AVA (Automated Variation Analysis) pipeline. A mutation report, memo, Nanodrop data, chip images, and chromatograms displaying variations will all be sent electronically to you through the LMT Molecular Diagnostics Wizard.