Date of Award

5-2007

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Jeffrey Becker

Committee Members

Pamela Small, David Joy, Steven Wilhelm

Abstract

The inauguration of the International Human Genome Initiative in the later part of the 1980’s coincided with the development of scanning probe microscopy (SPM). SPM was a good fit as one of the new technologies that might be implemented to sequence or map DNA and perhaps make a major contribution toward the goal of sequencing the entire human genome. Although the scanning tunneling microscope (STM) was invented in 1982 [Binnig 1982] and the atomic force microscope (AFM) in 1986 [Binnig 1986], it was not until 1987 that the first STM became commercially available; the AFM became available in 1989.

Our group entered this exciting new scientific adventure in genome research towards the end of 1987. An interdisciplinary team was assembled that capitalized on our expertise in the fabrication and use of scanning tunneling microscopes. Additionally, we purchased one of the first commercially available scanning tunneling microscopes. Rounding out the team was expertise in imaging biomolecules such as DNA using electron microscopy.

Our initial research focused on STM imaging and scanning tunneling spectroscopy (STS) of 1) tobacco mosaic virus adsorbed to gold surfaces [Mantovani 1990] and 2) DNA passively mounted on highly ordered pyrolytic graphite (HOPG) surfaces [Allison 1990]. Our spectroscopy results with DNA encouraged speculation that the electronic signatures of nucleotide bases might be used to sequence DNA. However, in this early work we also discovered that simply adsorbing either virus or DNA to gold or HOPG surfaces resulted in substantial amounts of the sample being removed by the STM tunneling tip.

In order to more firmly immobilize negatively charged DNA molecules onto surfaces for STM scanning, we created gold sample surfaces with positive functionality mediated by a self-assembled monolayer of 2-dimethylaminoethane thiol. Using this method, we produced the first reported images of entire genetically functional plasmid DNA molecules obtained by STM [Allison 1992a, Allison 1992b Allison 1993, Bottomley 1992].

Efforts initiated by our laboratory to restriction map DNA molecules by AFM imaging were successful. This was accomplished by physically mapping the location of a mutant EcoRI endonuclease that binds to but does not cleave large DNA clones. Our new AFM technology was pioneered as an alternative to conventional gel-based restriction mapping; it was first demonstrated on plasmid DNA molecules [Allison 1996] and later on larger molecules including cosmid clones [Allison 1997]. This technology should prove to be more effective than conventional mapping methods because by using AFM mapping neither the number nor the proximity of restriction sites to one another is problematic.

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