CORE--INSTRUMENTATION AND MUTATION DETECTION

Weill Medical College Of Cornell Univ, New York NY

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Abstract

Identifying genetic and epigenetic changes associated with tumor development and progression will require high-throughput detection methods. The goal of this core is to provide instrumentation and mutation detection support to achieve large scale analysis of mutations, deletions, insertions, LOH, gene amplifications, methylation status and gene expression levels. At least two different and independent technologies will be used to score critical changes. This core will have the following responsibilities: (i) Provide instrumentation for oligonucleotide synthesis of PCR primers, LDR primers, and DNA arrays. Core A will provide the large numbers of oligonucleotides necessary to fully develop the PCR/LDR/array based methods described in Project 1, (ii) Provide instrumentation for manufacture and analysis of DNA arrays. Core A will provide the universal DNA arrays and image analysis facilities required for Project 1; (iii) Provide for Taqman assays to determine gene expression and gene copy levels for RPTK's and candidate genes identified in Project 2, and (iv) Provide instrumentation for DNA sequencing and analysis of mutations associated with cancer. Core A will provide equipment for PCR amplification, LDR reactions, dideoxy sequencing, and EndoV/DNA ligase mutation scanning. To assure that the Aims of Project 1 are completed, the revision includes validation with microsatellites for LOH analysis and with Taqman analysis for gene copy number and expression levels. Thus, we aim to use at least two different approaches to confirm copy changes. As demonstrated with our p53 mutation detection work, multiple approaches to determine mutational status provided the most accurate results. Likewise, multiple independent determinations of copy number levels (both at the RNA and DNA level) will provide the most accurate assessment of gene status.