Presentation Title

Gibson Assembly of PCR-Produced DNA Fragments

Format of Presentation

Poster to be presented the Friday of the conference

Presenter Information

Brandon A. BasslerFollow

Abstract

In molecular biology, the polymerase chain reaction (PCR) is a tool that allows for the selective amplification of any specific target DNA sequence and is widely used in the construction of recombinant DNA molecules. Joining together PCR-produced DNA fragments to form such molecules is often a difficult process, as PCR amplification produces DNA with "blunt" ends; this causes the classical method of using DNA ligase alone to become unreliable as the DNA fragments lack the complementary (or "sticky") ends that bond spontaneously in solution and increase the efficacy of DNA ligase significantly. This project plans to explore the use of a newer method of joining blunt-ended fragments known as Gibson assembly, which uses DNA ligase in conjunction with DNA polymerase and exonuclease in an isothermal sequence of reactions to assemble fragments. The only additional requirement of Gibson assembly is that the fragments must have overlapping sequences at the end where they join, as this results in sticky ends when the ends of the five-prime strands undergo degradation by the exonuclease in the first reaction. The exonuclease then denatures from the heat, and the degradation in the now-annealed strands is repaired by DNA polymerase. DNA ligase can then join the adjacent ends of the strands. In theory, Gibson assembly accomplishes in an hour what would otherwise take days or weeks using classical methods; the aim of this project is to directly compare the results of Gibson assembly to traditional methods of ligating PCR-produced DNA fragments.

Department

Biological Sciences

Faculty Advisor

Donald Nelson

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Gibson Assembly of PCR-Produced DNA Fragments

In molecular biology, the polymerase chain reaction (PCR) is a tool that allows for the selective amplification of any specific target DNA sequence and is widely used in the construction of recombinant DNA molecules. Joining together PCR-produced DNA fragments to form such molecules is often a difficult process, as PCR amplification produces DNA with "blunt" ends; this causes the classical method of using DNA ligase alone to become unreliable as the DNA fragments lack the complementary (or "sticky") ends that bond spontaneously in solution and increase the efficacy of DNA ligase significantly. This project plans to explore the use of a newer method of joining blunt-ended fragments known as Gibson assembly, which uses DNA ligase in conjunction with DNA polymerase and exonuclease in an isothermal sequence of reactions to assemble fragments. The only additional requirement of Gibson assembly is that the fragments must have overlapping sequences at the end where they join, as this results in sticky ends when the ends of the five-prime strands undergo degradation by the exonuclease in the first reaction. The exonuclease then denatures from the heat, and the degradation in the now-annealed strands is repaired by DNA polymerase. DNA ligase can then join the adjacent ends of the strands. In theory, Gibson assembly accomplishes in an hour what would otherwise take days or weeks using classical methods; the aim of this project is to directly compare the results of Gibson assembly to traditional methods of ligating PCR-produced DNA fragments.