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Uniform TitleQuantitative analysis of genetic variations using molecular affinity and MALDI-TOF mass spectrometry
NameKim, Aana Moon (author), Kim, Sobin (chair), Chabal, Yves (internal member), Cai, Li (internal member), Rutgers University, Graduate School-New Brunswick,
Degree Date2007
Date Created2007
SubjectBiomedical Engineering,
DNA,
Time-of-flight mass spectrometry
DescriptionA system for DNA quantification is demonstrated utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and a molecular affinity system. MALDI-TOF MS can detect nucleic acids in milliseconds with high resolution, which allows for a high throughput sample analysis. The resulting mass spectra can produce more accurate results than conventional methods based on gel electrophoresis especially when secondary structures exist in the sample DNA molecules. Secondary DNA structures can lead to incomplete separation in the gel and reduce the accuracy in DNA sequence determination. MALDI-TOF MS measures the intrinsic property of DNA mass, and thus allows unambiguous detection of DNA fragments. MALDI TOF MS can be further facilitated by molecular affinity systems such as biotin-streptavidin as they increase the accuracy and throughput of MS.
Our approach is based on SPC-SBE, a previously developed method for high throughput genotyping employing molecular affinity, and involves optimization in both sample preparation and MALDI-TOF MS spectral analysis by signal processing. Creating small sample spots with a stainless steel needle to reduce sample crystal heterogeneity improved the DNA quantification system. To further reduce the impact of salt adduct peaks, signal-processing techniques were used for better signal separation. A basic signal processing technique involved ensemble average and ensemble standard deviations of spectra. Ensemble averaging is a simple and powerful signal processing technique to reduce noise. Additionally, the microbead pipette tip device was tested as a new method to rapidly isolate biotinylated DNA fragments and increase throughput of the molecular affinity system. We demonstrate our method provides high quantification accuracy, and therefore can be efficiently used for the application of rapid high-throughput gene expression monitoring.
Our approach is based on SPC-SBE, a previously developed method for high throughput genotyping employing molecular affinity, and involves optimization in both sample preparation and MALDI-TOF MS spectral analysis by signal processing. Creating small sample spots with a stainless steel needle to reduce sample crystal heterogeneity improved the DNA quantification system. To further reduce the impact of salt adduct peaks, signal-processing techniques were used for better signal separation. A basic signal processing technique involved ensemble average and ensemble standard deviations of spectra. Ensemble averaging is a simple and powerful signal processing technique to reduce noise. Additionally, the microbead pipette tip device was tested as a new method to rapidly isolate biotinylated DNA fragments and increase throughput of the molecular affinity system. We demonstrate our method provides high quantification accuracy, and therefore can be efficiently used for the application of rapid high-throughput gene expression monitoring.
Note[degree] M.S.
Note[bibliography] Includes bibliographical references (p. 23-24).
Genretheses
Persistent URLhttp://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.13469
LanguageEnglish
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.