Advances in single-molecule nucleic acid sequencing

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 att...

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Main Author: Lacenere, Christopher J.
Format: Others
Published: 2006
Online Access:https://thesis.library.caltech.edu/5252/1/thesis-c.lacenere.pdf
Lacenere, Christopher J. (2006) Advances in single-molecule nucleic acid sequencing. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/CA4B-S389. https://resolver.caltech.edu/CaltechETD:etd-09302006-140602 <https://resolver.caltech.edu/CaltechETD:etd-09302006-140602>
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spelling ndltd-CALTECH-oai-thesis.library.caltech.edu-52522019-12-21T03:05:28Z Advances in single-molecule nucleic acid sequencing Lacenere, Christopher J. 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. 2006 Thesis NonPeerReviewed application/pdf https://thesis.library.caltech.edu/5252/1/thesis-c.lacenere.pdf https://resolver.caltech.edu/CaltechETD:etd-09302006-140602 Lacenere, Christopher J. (2006) Advances in single-molecule nucleic acid sequencing. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/CA4B-S389. https://resolver.caltech.edu/CaltechETD:etd-09302006-140602 <https://resolver.caltech.edu/CaltechETD:etd-09302006-140602> https://thesis.library.caltech.edu/5252/
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sources NDLTD
description 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.
author Lacenere, Christopher J.
spellingShingle Lacenere, Christopher J.
Advances in single-molecule nucleic acid sequencing
author_facet Lacenere, Christopher J.
author_sort Lacenere, Christopher J.
title Advances in single-molecule nucleic acid sequencing
title_short Advances in single-molecule nucleic acid sequencing
title_full Advances in single-molecule nucleic acid sequencing
title_fullStr Advances in single-molecule nucleic acid sequencing
title_full_unstemmed Advances in single-molecule nucleic acid sequencing
title_sort advances in single-molecule nucleic acid sequencing
publishDate 2006
url https://thesis.library.caltech.edu/5252/1/thesis-c.lacenere.pdf
Lacenere, Christopher J. (2006) Advances in single-molecule nucleic acid sequencing. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/CA4B-S389. https://resolver.caltech.edu/CaltechETD:etd-09302006-140602 <https://resolver.caltech.edu/CaltechETD:etd-09302006-140602>
work_keys_str_mv AT lacenerechristopherj advancesinsinglemoleculenucleicacidsequencing
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