Abstracts: 

 SBIR-Phase I 



Improvements in DNA Sequencing Methods by 

 Incorporating Chemiluminescent Detection 



John C. Vovta 



Tropix. Inc.. Bedford. MA 01730 

 (617) 271-0045 



The research plan for Phase I of this project will involve feasibility studies of the use of 

 chemiluminescence in DNA sequencing. With the advent of enzymatically induced 

 chemiluminescence from stable precursors and the improved instrumentation for 

 luminometry. ultrasensitive detection of a wide variety of agents under many different 

 conditions is now possible. With the implementation of the chemiluminescent substrate 

 technology, we believe that it will be possible to improve several aspects of DNA 

 sequencing methodology such as stabilizing reagents, eliminating radioisotopic labels, 

 shortening the incubation times, improving detection sensitivities, and greatly 

 simplifying the detection instrumentation. To verify the viability of such a system, we 

 plan to use an avidin/biotin detection system with alkaline phosphatase as the signal- 

 generating label. In one study, biotinylated primer strands and other necessary reagents 

 will be used in the dideoxy-sequencing reactions and separated via gel electrophoresis. 

 Subsequently, the separated DNA will be visualized with avidin-labeled alkaline 

 phosphatase and a chemiluminescent substrate. The use of chemiluminescent substrates 

 in multiplex-sequencing protocols will also be investigated. In Phase I, the luminescent 

 detection of nucleotides will be accomplished with X-ray and instant films. The 

 anticipated improvements in chemiluminescence-based DNA sequencing will be fully 

 explored in Phase II with the implementation of full -performance protocols coupled 

 with the design and prototype generation of a simple and inexpensive luminescent gel/ 

 blot scanner capable of high throughput. Fmally. we believe that these new and 

 improved sequencers will offer speed, cost effectiveness, and ease of use in DNA 

 sequence determinations. 



