Lacenere, Christopher J. (2006) Advances in single-molecule nucleic acid sequencing. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-09302006-140602
The ability to quickly and accurately obtain sequence information from single molecules of DNA and RNA has far-reaching implications for our understanding of biology. In the work presented here, we have made several advances in the area of single-molecule DNA and RNA sequencing. Specifically, in attempting to increase the read length of DNA polymerase, we have assayed several custom synthesized fluorescent nucleotides containing longer dye–base linkers. We have validated the efficacy of these nucleotides at both bulk and single-molecule levels. Furthermore, we have screened several commercially available DNA polymerases for their ability to incorporate these nucleotides. We also show that reverse transcriptase is able to synthesize a complimentary DNA strand of 28 bases in length from an RNA template, using solely fluorescently labeled nucleotides. Additionally, we show that reverse transcriptase is able to incorporate a fluorescently labeled nucleotide into an RNA template at the single-molecule level. Finally, we demonstrate automated reagent exchange for our single-molecule sequencing system.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||DNA; nucleic acids; nucleotides; polymerase; reverse transcriptase; RNA; sequencing; single-molecule|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||22 July 2005|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||02 Oct 2006|
|Last Modified:||26 Dec 2012 03:16|
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