) Development of cost-effective, high-throughput sampling coupled to rapid, parallel DNA profiling methods will facilitate molecular analysis of normal and diseased states. This R21/R33 initiative focuses on detection of single nucleotide polymorphisms (SNPs) and point mutations in cancer. Our goals are as follows: Milestones/Aims for the R21 phase include: 1) Development of a robust, 4-color rhodamine BigDye terminators-based single nucleotide extension (SNE) array assay which will allow DNA TaqFS-catalyzed, single nucleotide extension to detect a wide variety of SNPs/mutations associated with human cancer. We will increase signal intensity as well as eliminate the need for synthesis of costly spacers on array-bound probes by using dendrimer-coated surfaces; and, 2) Scale-up the assay to simultaneously monitor 30-50 SNPs/mutations on a single array. Multiplexing will be done both at PCR and SNE steps and use of total genomic DNA targets will be tested. Strategies will also be implemented to screen multiple individuals on a single chip for the same mutation.
Aims for the R33 phase include: 1) Application of SNE assay to type SNPs within and flanking the minimal deleted region in lp, llq and 14q in neuroblastoma pediatric patients. The assay will facilitate identification of deletion location and extent and eventually will facilitate understanding the relationship between genotype and phenotype; and 2) Identification of single-base changes occurring within the mutation cluster region of adenomatous polyposis coli (APC) gene in patients with familial adenomatous polyposis (FAP) and in sporadic colorectal cancer. We will also evaluate genotype/phenotype relationships using information from SNE-based mutation analyses and results from array-based genome-wide mRNA expression profiles already funded by an NCI Contract. Array-based results generated from Aims 1 and 2 will be compared and contrasted to data available from previously typed individuals using more traditional DNA analysis approaches as well as to solution-phase results generated at PE-Biosystems. Development and validation of array-bound approaches for monitoring DNA changes in cancer should facilitate high throughput, parallel processing of samples for critical human cancer genes. In addition, it will help establish basic knowledge required to rapidly define the molecular basis of specific malignancies and eventually provide a foundation for rational molecular assessment of therapeutic.
