The purpose of this proposal is to develop a rapid, analytical, and cost- effective technology for molecular diagnostics and high-throughput DNA analysis. Our innovative approach is based on interfacing a polymerase chain reaction (PCR) method with DNA analysis using infrared (IR) matrix- assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Future innovations to this method, including the development of new matrices, the investigation of desorption at various wavelengths, and the refinement of extraction techniques (such as delayed extraction or time-lag focusing) will improve its performance and increase its utility for the genotyping of DNA. In Phase I we will utilize a high performance IR- MALDI-MS for the analysis of trinucleotide expansion in the neurodegenerative disorders, such as Huntington's disease, DRPLA and in the MAB21L1 gene at 13q13. We will design and validate disease-specific primer pairs for the generation of a smaller amplicons for the efficient detection of expanded alleles by MALDI-MS. We will develop a protocol for rapid purification of the PCR product and a matrix suitable for MALDI at lR to enable rapid DNA genotyping. This approach represents a refinement to the current methods of analysis by conventional gel electrophoresis.
The analysis of genes containing CAG trinucleotide repeats & microsatellite markers.Many medical and clinical applications,such as analysis of DNA & RNA, proteins and peptides, The diode-pumped ultra-short pulse Gr:LiSAF will make the mass spectrometer, compact, affordable and rugged, enabling commercialization of cost effective, MALDI-MS analysis in diagnostic, industrial lab, health-care, medicine, agriculture, and environmental monitoring fields.
