(603f) Molecular Affinity System for High Throughput Monitoring of Allele-Specific Gene Expression by MALDI-TOF MS

A novel mass spectrometry based method, Solid Phase Capture-Single Base Extension (SPC-SBE), allows the analysis of larger numbers of variations than other mass spectrometry based techniques. It involves extension of a library of SBE primers by a single base at the SNP site using biotinylated terminators and isolation of biotinylated fragments on a streptavidin-coated surface by molecular affinity. Isolated extension products are analysed by mass spectrometry and difference in mass between extension products and corresponding primers is used for inferring base identities. It has been shown that up to 50 sequence variations such as SNPs can be genotyped simultaneously using the method. Recently, we have employed the approach to develop a multiplexed method that will efficiently allow allele-specific gene expression analysis. In this method, the high multiplexing capacity of SPC-SBE combined with facilitated sample purification due to the SPC resulted in effective measurement of allele-specific determination of multiple mRNAs. However, current implementation of SPC-SBE is still limited in its capacity for processing multiple samples rapidly. We report here the fabrication of a microbead device that allows rapid isolation of biotinylated fragments, which can be released by a change in pH and directly spotted on a MALDI sample plate for gene expression analysis. We have used the device for concurrent, allele-specific analysis of multiple gene transcripts. This demonstrates preliminary feasibility of the device and potential for its implementation in an automated format.