This project proposes to demonstrate the feasibility of a novel multiplexed chemiluminescent system for manual DNA sequencing or detection of double-stranded DNA that can potentially replace the use of radioprobes in small scale sequencing applications. the multiplexed detection is achieved using two enzyme labels and two chemiluminescent reagents to allow rapid, consecutive detection of two different enzyme-labeled probes on the same lane of the blotting membrane. This will allow sequence analysis to be carried out in two lanes, instead of requiring four as is the case in radiolabeling. Specially, the analysis is carried out by using primers that have specific haptens labeled onto them. After electrophoresis is carried out, the electrophoresis bands are transferred to a nylon membrane and exposed to a binding agent to the hapten that contains either HRP or AP. After rinsing, reagents are added to develop the chemiluminescent reaction for HRP using the chromophobe, Lumigen PS-3 (developed in house). After the image of the gel is acquired, this reaction is quenched by adding excess hydrogen peroxide and high pH. Then, the reagents for the AP reaction are added, including the chromophobe for this chemiluminescent reaction, Lumigen PPD. This approach allows either detection of two different termination products per lane or two different templates per lane. In essence, the approach allows doubling the throughput of manual sequencing.
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