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Full-Text Articles in Life Sciences
Ex Vivo Dna Cloning, Adam B. Fisher
Ex Vivo Dna Cloning, Adam B. Fisher
Theses and Dissertations
Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are …
Atm-Dependent Erk Signaling In Response To Dna Double Strand Breaks, Ashraf Khalil
Atm-Dependent Erk Signaling In Response To Dna Double Strand Breaks, Ashraf Khalil
Theses and Dissertations
Ionizing radiation (IR) triggers many signaling pathways stemming from DNA damage, and, independently, from extra-nuclear events. To generate radio-mimetic DNA double-strand breaks (DSBs) without and minimizing the effects on extra-nuclear radiation targets, human (p53+) glioma and carcinoma cells containing bromodeoxyuridine (BrdU)- substituted DNA were treated with Hoechst 33258 followed by long wave-length UV (UV-A) (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose-dependent generation of DSBs equivalent to 0.2 - 20 Gy of IR, as detected by pulse-field gel electrophoresis, accompanied by dose-dependent H2AX phosphorylation at ser-139 and ATM phosphorylation at ser-1981, indicating ATM activation. Furthermore, BrdU photolysis increased phosphorylation of …