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Type of Document	Dissertation
Author	Lacenere, Christopher J.
URN	etd-09302006-140602
Persistent URL	http://resolver.caltech.edu/CaltechETD:etd-09302006-140602
Title	Advances in single-molecule nucleic acid sequencing
Degree	PhD
Option	Biology
Advisory Committee	Advisor Name Title Raymond Joseph Deshaies Committee Chair Brian M. Stoltz Committee Member Michael Elowitz Committee Member Stephen R. Quake Committee Member
Keywords	single-molecule sequencing RNA nucleotides nucleic acids DNA reverse transcriptase polymerase
Date of Defense	2005-07-22
Availability	unrestricted
Abstract 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.
Files	Filename       Size       Approximate Download Time (Hours:Minutes:Seconds)     28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access    thesis-c.lacenere.pdf 14.26 Mb 01:06:02 00:33:57 00:29:42 00:14:51 00:01:16