CORE--INSTRUMENTATION AND MUTATION DETECTION

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(Applicant's Description) Identifying multiple cancer-associated mutations will require high throughput detection methods. The goal of this core is to provide the instrumentation and mutation detection support to achieve large-scale detection and analysis of mutations. This core will have the following responsibilities: (1) Provide instrumentation for oligonucleotide synthesis and for analysis of mutations associated with cancer. Core A will provide the large numbers of oligonucleotides necessary to fully develop the PCR/LDR and PCR/RE/LDR methods described in Project 1. The products of these ligation detection reactions are currently separated and quantified on an ABI 373A DNA sequencer. In the future, LDR products may be simultaneously detected using addressable oligonucleotide or PNA zip-code arrays. (ii) Provide instrumentation for confirming the nature of cancer mutations and identification of additional thermophilic ligase genes by DNA sequencing. Automatic PCR-based sequencing technology will be applied both for assessing the reliability of PCR/LDR and determining the sequences of additional thermophilic ligase genes. (iii) Testing polymerase fidelity and the efficiency of nucleotide conversions using convertide oligonucleotides. The PCR/RE/LDR cancer detection method has the potential of detecting cancer mutations at a sensitivity of 1 in 100,000 to 1,000,000. The sensitivity of PCR/RE/LDR is dependent on the fidelity of polymerase extension from primers containing a 3' nucleotide analogue (Project 2). An assay has been developed to test both the relative efficiency and fidelity of different polymerases for each nucleotide conversion.