Enzyme Switch: many reporter molecules from a single-molecule-sequencing product

DESCRIPTION provided by applicant Even as the cost and throughput of commercial sequencers has continued to improve over the last years there is still a need to further reduce sequencing costs to increase throughput and sequencing accuracy and to reduce the costs associated with sample preparation Single molecule methods such as the Pacific Biosciences or nanopore technologies have the potential to reduce sample preparation bottlenecks but suffer from very high raw error rates We are developing the Activator Sequencing technology for single molecule sequencing with low error rates The method is applicable to a variety of read outs such as fluorescence luminescence pH sensing and electrochemistry many of which can be used in a disposable CMOS chip platform similar to that of Ion Torrent If successful Activator Sequencing would enable low cost long read length high accuracy sequencing on a scalable platform capable of leveraging semiconductor industry know how and investments to yield continued yearly increases in performance based on Mooreandapos s Law type decreases in feature size Activator Sequencing uses a andquot molecular amplifierandquot to convert the products of a single molecule sequencing reaction into many copies of a readily detectable reporter molecule Specifically sequencing by synthesis is performed using dNTPs labeled at the terminal phosphate with an enzyme activator Upon incorporation of a dNTP onto a primed template an activator is released which can turn an engineered enzyme switch from an andquot offandquot to an andquot on conformation Each activated enzyme can rapidly generate a multitude of detectable products thereby amplifying the detectable signal from the original dNTP incorporation For example while the Ion Torrent system needs many template copies to generate a detectable pH signal an activator released from a single dNTP molecule can turn on a single enzyme molecule to generate tens of thousands of protons in a few seconds The generation of multiple copies of a reporter makes it easier to detect nucleotide incorporation thereby allowing single molecule sequencing with low noise Such single molecule sequencing would simplify sample preparation and enable very long read lengths by eliminating dephasing limitations If combined with low cost highly parallel CMOS sensors instrumentation costs would be greatly reduced compared to fluorescence instrumentation Our preliminary results demonstrate that an engineered enzyme switch can function as such a andquot molecular amplifier andquot The proposed Phase I SBIR grant will demonstrate the ability of Activator Sequencing to use an engineered enzyme switch to perform single molecule sequencing with high accuracy using fluorescence detection Future work would focus on transferring the technology to a scalable integrated CMOS sensor PUBLIC HEALTH RELEVANCE Nucleic acid sequence information is critical to medical research and to basic biological studies The goal of this project is to demonstrate the feasibilit of DNA sequencing system which may be capable of de novo sequencing of mammalian genomes for under $ Such a platform will allow researchers and clinicians to perform the translational research ultimately required to improve human health