| In this research, biotinylated dideoxynucleotide terminators and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were combined to create (1) a mini-sequencing assay for determining DNA sequences and (2) a single base extension (SBE) assay to improve accuracy and increase multiplexed detection of single nucleotide polymorphisms (SNPs). Secondly, high-throughput sequencing technologies were used to conduct the first large-scale EST (expressed sequence tag) sequencing of the model organism Aplysia californica.; Compared to electrophoresis based DNA sequencing systems, MS produces fast separation on microsecond time scales and high resolution of sequencing fragments to accurately detect mutations and heterozygotes. However for accurate MS analysis, DNA fragments must be desalted and purified of contaminants. We have developed novel MS-based DNA sequencing and SBE methods to analyze DNA extension fragments in one tube using biotinylated dideoxynucleotides. The product fragments carrying a biotin at the 3′ end are purified of salts, false stops, and excess primers by capture with streptavidin coated magnetic beads. After capture and release, only correctly terminated biotinylated fragments are analyzed by MS to yield either accurate DNA sequencing data (sequencing method) or multiplexed genotyping data (SBE).; Since new protein synthesis is the primary difference between the formation of short- and long-term memory in Aplysia and higher organisms, a key to further understanding memory formation is the inventory and abundance of mRNA messages in the central nervous system. With its large, well-identified neurons and simple neuronal network, Aplysia has been a powerful model for elucidating the elementary mechanisms of synaptic transmission, and the pathways involved in short- and long-term plasticity and memory formation. Representing more than 20 times the number of sequences previously submitted to GenBank, 37,214 EST sequences were generated in this study from cDNA libraries from Aplysia's pedal-pleural ganglia. Among these transcripts were several with homology to neuron-specific and intracellular signaling peptides along with 9 homologs to human proteins associated with neurological disorders. These sequences can immediately be considered primary targets for future molecular studies in Aplysia. |
