Genome Analysis: Mutation Analysis Using Near Infrared Laser-Induced Fluorescence (NIR-LIF) and Single Molecule Detection in Microfluidic Devices

A number of genotyping methods have been developed for mutation analysis, each of which has its own unique advantage. DNA amplification via polymerase chain reaction (PCR) provides an unlimited supply of material for subsequent genetic analysis even in case where only a single copy of the DNA molecu...

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Main Author: Wabuyele, Musundi Ben
Other Authors: Erwin D. Poliakoff
Format: Others
Language:en
Published: LSU 2003
Subjects:
Online Access:http://etd.lsu.edu/docs/available/etd-0403103-111832/
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spelling ndltd-LSU-oai-etd.lsu.edu-etd-0403103-1118322013-01-07T22:48:25Z Genome Analysis: Mutation Analysis Using Near Infrared Laser-Induced Fluorescence (NIR-LIF) and Single Molecule Detection in Microfluidic Devices Wabuyele, Musundi Ben Chemistry A number of genotyping methods have been developed for mutation analysis, each of which has its own unique advantage. DNA amplification via polymerase chain reaction (PCR) provides an unlimited supply of material for subsequent genetic analysis even in case where only a single copy of the DNA molecule is present in the sample. Research presented in this dissertation first examines the efficiency of in vitro amplification of single copy DNA with subsequent sequencing analysis of PCR product. The PCR products (amplicons) were investigated for possible alteration, distortions or mutations due to the amplification process. The sequencing data for single copy amplification indicated a read length of 424 bases could be achieved with read accuracy of 99.3%. A confocal detection system was constructed to monitor the fluorescence signature from single DNA molecule labeled with near infrared (NIR) dyes. The performance of the system was first tested by applying single photon burst technique to detect double-stranded DNA molecules in polymeric devices. Sampling efficiencies were investigated by physically narrowing the channel sizes of the microdevices and by applying electrokinetic focusing. The narrow channels showed 4 times improvement in a 15 µm channel compared to a 50 µm channel. Similar results were demonstrated in the focusing studies. Single molecule sizing of ë-DNA, M13, pUC19 and pBR322 DNA using single photon burst was also demonstrated. In addition single molecule detection of a NIR chromophore NN383 was analyzed in PC devices and a detection efficiency of 94% was achieved. The other application for the NIR system was in a new strategy for analyzing molecular signatures of disease states in real-time using single-pair fluorescence energy transfer (spFRET) coupled with ligase detection reaction (LDR) to rapidly detect single base mutations in codon 12 of K-ras oncogene which has high diagnostic value for colorectal cancer. LDR-spFRET provided the necessary specificity and sensitivity to detect single point mutations in as little 600 copies of human genomic DNA without PCR amplification at a level of 1 in 1000 wildtype sequences. In addition the assay demonstrated analysis times < 5 min. Erwin D. Poliakoff Gregg Pettis Robert L. Cook Paul S. Russo Soper, Steven A. LSU 2003-04-04 text application/pdf http://etd.lsu.edu/docs/available/etd-0403103-111832/ http://etd.lsu.edu/docs/available/etd-0403103-111832/ en unrestricted I hereby grant to LSU or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University Libraries in all forms of media, now or hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.
collection NDLTD
language en
format Others
sources NDLTD
topic Chemistry
spellingShingle Chemistry
Wabuyele, Musundi Ben
Genome Analysis: Mutation Analysis Using Near Infrared Laser-Induced Fluorescence (NIR-LIF) and Single Molecule Detection in Microfluidic Devices
description A number of genotyping methods have been developed for mutation analysis, each of which has its own unique advantage. DNA amplification via polymerase chain reaction (PCR) provides an unlimited supply of material for subsequent genetic analysis even in case where only a single copy of the DNA molecule is present in the sample. Research presented in this dissertation first examines the efficiency of in vitro amplification of single copy DNA with subsequent sequencing analysis of PCR product. The PCR products (amplicons) were investigated for possible alteration, distortions or mutations due to the amplification process. The sequencing data for single copy amplification indicated a read length of 424 bases could be achieved with read accuracy of 99.3%. A confocal detection system was constructed to monitor the fluorescence signature from single DNA molecule labeled with near infrared (NIR) dyes. The performance of the system was first tested by applying single photon burst technique to detect double-stranded DNA molecules in polymeric devices. Sampling efficiencies were investigated by physically narrowing the channel sizes of the microdevices and by applying electrokinetic focusing. The narrow channels showed 4 times improvement in a 15 µm channel compared to a 50 µm channel. Similar results were demonstrated in the focusing studies. Single molecule sizing of ë-DNA, M13, pUC19 and pBR322 DNA using single photon burst was also demonstrated. In addition single molecule detection of a NIR chromophore NN383 was analyzed in PC devices and a detection efficiency of 94% was achieved. The other application for the NIR system was in a new strategy for analyzing molecular signatures of disease states in real-time using single-pair fluorescence energy transfer (spFRET) coupled with ligase detection reaction (LDR) to rapidly detect single base mutations in codon 12 of K-ras oncogene which has high diagnostic value for colorectal cancer. LDR-spFRET provided the necessary specificity and sensitivity to detect single point mutations in as little 600 copies of human genomic DNA without PCR amplification at a level of 1 in 1000 wildtype sequences. In addition the assay demonstrated analysis times < 5 min.
author2 Erwin D. Poliakoff
author_facet Erwin D. Poliakoff
Wabuyele, Musundi Ben
author Wabuyele, Musundi Ben
author_sort Wabuyele, Musundi Ben
title Genome Analysis: Mutation Analysis Using Near Infrared Laser-Induced Fluorescence (NIR-LIF) and Single Molecule Detection in Microfluidic Devices
title_short Genome Analysis: Mutation Analysis Using Near Infrared Laser-Induced Fluorescence (NIR-LIF) and Single Molecule Detection in Microfluidic Devices
title_full Genome Analysis: Mutation Analysis Using Near Infrared Laser-Induced Fluorescence (NIR-LIF) and Single Molecule Detection in Microfluidic Devices
title_fullStr Genome Analysis: Mutation Analysis Using Near Infrared Laser-Induced Fluorescence (NIR-LIF) and Single Molecule Detection in Microfluidic Devices
title_full_unstemmed Genome Analysis: Mutation Analysis Using Near Infrared Laser-Induced Fluorescence (NIR-LIF) and Single Molecule Detection in Microfluidic Devices
title_sort genome analysis: mutation analysis using near infrared laser-induced fluorescence (nir-lif) and single molecule detection in microfluidic devices
publisher LSU
publishDate 2003
url http://etd.lsu.edu/docs/available/etd-0403103-111832/
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