SBIR-STTR Award

The objective of this study is to improve the efficiencyof performance of a nonisotopic detection method for DNAsequencing. A chemiluminescent detection procedure using1,2-dioxetane substrates for alkaline phosphatase has beendeveloped. With this method, DNA sequencing reaction productslabeled with biotinylated primers are imaged followingelectropboresis by transfer of the separated DNA fragments to nylonmembrane, with 1,2-dioxetane substrates. Upon dephosphorylation,these enzymatic substrates decompose and emit light. In Phase I,the feasibility of increasing the amount of DNA sequenceinformation that can be imaged from one membrane will be assessedby separating multiple sets of DNA sequencing reactions in eachlane of the gel, transferring the DNA to nylon membrane, anddetecting each set of sequencing reactions individually. This isaccomplished by utilizing multiple DNA sequencing primers for theDNA sequencing reactions. The primer for each reaction willcontain its own unique label. Each set of DNA sequencing reactionsis detected with an alkaline phosphatase conjugate specific for thelabel. The performance of various labels and enzyme conjugates isevaluated. The goal of the study is the development of optimumprocedures for rapid detection of each set of labeled DNAfragments. If the research is successful, newly developedprotocols and the individual reagents will be incorporated into aresearch kit that will become a useful tool for molecularbiologists for DNA sequencing.Anticipated Results/Potential Commercial Applications as described by the awardee:DNA sequencing kits will be developed for use withseveral labeled primers that will permit sequential imaging ofmultiple sets of DNA sequencing reactions with chemiluminescencefrom 1,2 dioxetane substrates.

The objective of this project is to develop research kits forperforming DNA sequencing and PCR analysis utilizing a multiplexlabeling approach and chemiluminescent detection. Phase I demonstrated the feasibility of increasing the amount ofDNA sequence information which can be determined from onemembrane by separating multiple sets of DNA sequencing reactionsin each gel lane, transferring the DNA to nylon membrane, anddetecting each set of sequencing reactions individually with 1,2-dioxetane substrates for alkaline phosphatase. This wasaccomplished by utilizing multiple DNA sequencing primers, eachwith its own unique label, and detecting each set of DNAsequencing reactions sequentially with alkaline phosphataseconjugates specific for each label. Various labels and enzymeconjugates were evaluated for performance. Phase 11 will furtheroptimize the procedure for the sequential detection of each setof labeled fragments and will incorporate the newly developedprotocols and the individual reagents into a research kit whichwill become a useful tool for molecular biologists, DNAsequencing, and PCR product analysis. A DNA sequencing reactionkit with multiple hapten labeled primers and a complementarychemiluminescent detection kit containing hapten specific enzymeconjugates will be produced. The chemiluminescent detection kitwill also be useful for the sequential identification ofoverlapping PCR products. The creation of a specific membraneoptimized for chemiluminescence will be investigated. Inaddition, an apparatus for automating the blot development stepswill be utilized, increasing throughput and permitting systemscale up. Techniques for interfacing the multiplex labeling DNAsequencing procedures with PCR amplification and single strandedDNA template purification will also be developed. Protocols forthese techniques will be incorporated into the research kits andwill greatly expand their utility.Anticipated Results/Potential Commercial Applications as described by the awardee:DNA sequencing is an integral part ofmany research programs, and has until recently been performedsolely with the use of radioactive isotopes. Environmental andsafety concerns demand the development of more efficient andnon-radioactive methods for the DNA sequence determination. Research kits will be developed for performing DNA sequencing andPCR analysis with multiplex labeling and chemiluminescentdetection using 1,2-dioxetane substrates for alkalinephosphatase. Membranes which exhibit optimum signal to noisecharacteristics when performing multiple chemiluminescentdetections will be produced.