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 whose 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 oligonucleotide, 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 the 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 triphosphate, 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 standard (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 evaluated 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 increasing 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 human 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. ? ? ?
|Hutter, Daniel; Kim, Myong-Jung; Karalkar, Nilesh et al. (2010) Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups. Nucleosides Nucleotides Nucleic Acids 29:879-95|
|Chen, Fei; Gaucher, Eric A; Leal, Nicole A et al. (2010) Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection. Proc Natl Acad Sci U S A 107:1948-53|