This project will create an automated flow process polymerase chain reaction/cycle sequencer (PCR/CS) instrument to prepare DNA sequencing reactions and inject them into a capillary array instrument for analysis. The PCR\CS instrument will simultaneously perform 96 PCR and Sanger cycle sequencing reactions with fluorescently labeled primers in nanoliterscale reaction volumes. It will be part of an integrated DNA sequencing (DNASEQTM) system that will automate all steps from clone picking to the production of finished sequence information. The PCR/CS instrument will be a computer-controlled microfluidics flow-through system that uses arrays of capillaries for thermal cycling of PCR and cycle sequencing reactions; the thermal cycling will be performed with large-scale pumps recirculating water. In the phase I, we will prove the concept by building a single- channel PCR/CS instrument using 75 mum inside diameter (I.D.) capillary loops for the thermal cycling processes, Reagent Injection Ports for adding reactants, and microvalves. We will also modify the injection manifold of the Molecular Dynamics DNA sequencer, the capillary array instrument, to allow us to analyze nanoliter volumes of labeled DNA fragments. In phases II and III, we will build multi-channel prototypes, miniaturize the devise to smaller I.D. capillaries, and integrate it into the DNASEQTM system.Proposed commercial application:The project will develop an automated flow process for sample preparation and DNA sequencing. The market for the PCR/CS instrument will be genome centers, pharmaceutical and biotechnology companies, universities, and genetic diagnostic laboratories.National Center for Human Genome Research (NCHGR)
