Multipurpose reversible terminators
Small Business Information
FIREBIRD BIOMOLECULAR SCIENCES, LLC, PO BOX 13983, GAINESVILLE, FL, 32641
AbstractDESCRIPTION (provided by applicant): The proposed research will transfer technology from the Foundation for Applied Molecular Evolution (FfAME) to Firebird Biomolecular Sciences LLC, allowing Firebird to commercialize multi-use nucleoside triphosphates who se 3'-OH groups are blocked by a NH2 group. These triphosphates should be accepted by terminal transferase, Terminator DNA polymerase, HIV reverse transcriptase, and mutants of Taq DNA polymerase. When incorporated at the 3'-end of a growing oligonucleotid e, the NH2 group blocks extension (just as a 2',3'-dideoxynucleotide does). Unlike a dideoxynucleotide, however, this blockage is reversible; application of a simple, mild reagent rapidly but mildly cleaves the 3'-O-NH2 bond to regenerate the natural 3'-OH group, yielding natural DNA again. Unlike other such terminators, however, the 3'-O-NH2 group not only blocks polymerase extension but also offers a handle that allows the terminated DNA to selectively react with certain functional groups, allowing for th e recovery of the extended, terminated DNA from complex biological mixtures, or SNP detection, among others. To enable this commercialization, we shall: (a) Synthesize the 3'-O-NH2 derivatives of thymidine triphosphate, 2'-deoxycytidine triphosphat e, 2'-deoxyadenosine triphosphate, and 2'-deoxyguanosine triphosphate. (b) Demonstrate their incorporation using the mutant Taq polymerase that supported the incorporation of TTP-O-NH2. (c) Evaluate the fidelity of incorporation in competition with standar d (3'-OH) and 2',3'- dideoxynucleoside triphosphates. (d) Synthesize a thymidine 3'-O-NH2 analog carrying a 5-position fluorescent group and demonstrate its incorporation using polymerases. Repeating the cycle of incorporation and cleavage supports sequencing using cyclic reversible termination (SuCRT), a sequencing strategy that can be highly multiplexed. Under confidentiality agreements with Intelligent Bio-Systems, Helicos, and others, this terminator-polymerase combination is presently being e valuated for their instruments and their sequencing architectures. Single cycles of reversible termination also have value. They permit the detection of single nucleotide polymorphisms (SNPs) and multinucleotide polymorphisms in architectures that have inc reasing complexity and increasing risk. Therefore, these terminators have multiple potential applications in a variety of research settings, and in architectures that might see involvement in human diagnostics. Therefore, they will be added to the growing portfolio of nucleic acid analogs offered by Firebird for sale, and underline the next phase in the development of Firebird as a corporation. Over the past century, our ability to connect genomic information to the molecular characterization of huma n tissues, and therefore connect molecules to normal and diseased biological function, has been driven by new chemistries that enable new analytical architectures because they deliver new specs . The proposed research will facilitate the commercialization of one of those new chemistries, which allows DNA primer extension to be reversibly terminated by a group that also can serve as a temporary handle to capture and analyze the extended product.
* information listed above is at the time of submission.