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Analysis of single nucleotide polymorphisms using molecular affinity separation and mass spectrometry

Descriptive

TypeOfResource
Text
TitleInfo (ID = T-1)
Title
Analysis of single nucleotide polymorphisms using molecular affinity separation and mass spectrometry
Identifier
ETD_1751
Identifier (type = hdl)
http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000051381
Language
LanguageTerm (authority = ISO639-2); (type = code)
eng
Genre (authority = marcgt)
theses
Subject (ID = SBJ-1); (authority = RUETD)
Topic
Chemical and Biochemical Engineering
Subject (ID = SBJ-1); (authority = ETD-LCSH)
Topic
Genetic polymorphisms
Abstract
Single nucleotide polymorphisms (SNP) constitute the most abundant human genetic variations and are important markers for studying interindividual variability with many different techniques having being developed for their study. The use of mass spectrometry (MS) is an attractive detection method for SNP genotyping due to label free detection based on molecular mass, especially for use in diagnostic applications that would require analysis of tens of SNPs in cohorts of individuals. The work in this thesis explores the use of a molecular affinity purification system for improving multiplexing levels of SNP genotyping using MS based detection and approaches for improving its throughput.
The strong molecular affinity between biotin and streptavidin has been employed for isolation of biotinylated oligonucleotides before analysis by MS using the previously developed solid phase capture-single base extension approach. We have been able to genotype up to 50 SNPs simultaneously using two genes from the cytochrome 450 family of genes as model system. These results have demonstrated the utility of the biotin-streptavidin affinity system for highly multiplexed SNP genotyping using MS. Following this, two approaches have been employed to reduce processing time and improve throughput of the technique. First, we have used monomeric avidin coated microbeads to fabricate a device that leads to a substantial reduction in processing time for the isolation step to ~2 hours, and allows simultaneous processing of multiple samples for genotyping a limited number of SNPs. Additionally, the microbead device can be reused 5 times with a simple regeneration protocol thus acting as a low cost tool for enhancing sample cleanup prior to MS. A second approach involves the use of heat and water for breaking the biotin-streptavidin interaction that allows direct analysis of released fragments by MS. We have shown its utility for reducing processing time after the isolation step substantially, and used it for highly multiplexed SNP genotyping. In concert, these studies demonstrate the feasibility of using the molecular affinity interaction between biotin and (strept)avidin for high throughput genotyping of single nucleotide polymorphisms using MS.
PhysicalDescription
Form (authority = gmd)
electronic resource
Extent
xi, 79 p. : ill.
InternetMediaType
application/pdf
InternetMediaType
text/xml
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references (p. 73-78)
Note (type = statement of responsibility)
by Ashish Misra
Name (ID = NAME-1); (type = personal)
NamePart (type = family)
Misra
NamePart (type = given)
Ashish
NamePart (type = date)
1981
Role
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author
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Ashish Misra
Name (ID = NAME-2); (type = personal)
NamePart (type = family)
Kim
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Sobin
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chair
Affiliation
Advisory Committee
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Sobin Kim
Name (ID = NAME-3); (type = personal)
NamePart (type = family)
Roth
NamePart (type = given)
Charles
Role
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internal member
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Advisory Committee
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Charles Roth
Name (ID = NAME-4); (type = personal)
NamePart (type = family)
Pedersen
NamePart (type = given)
Henrik
Role
RoleTerm (authority = RULIB); (type = )
internal member
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Advisory Committee
DisplayForm
Henrik Pedersen
Name (ID = NAME-5); (type = personal)
NamePart (type = family)
Chiew
NamePart (type = given)
Yee
Role
RoleTerm (authority = RULIB); (type = )
internal member
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Advisory Committee
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Yee Chiew
Name (ID = NAME-6); (type = personal)
NamePart (type = family)
Lee
NamePart (type = given)
KiBum
Role
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outside member
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Advisory Committee
DisplayForm
KiBum Lee
Name (ID = NAME-1); (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB); (type = )
degree grantor
Name (ID = NAME-2); (type = corporate)
NamePart
Graduate School - New Brunswick
Role
RoleTerm (authority = RULIB); (type = )
school
OriginInfo
DateCreated (point = ); (qualifier = exact)
2009
DateOther (qualifier = exact); (type = degree)
2009-05
Place
PlaceTerm (type = code)
xx
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
RelatedItem (type = host)
TitleInfo
Title
Graduate School - New Brunswick Electronic Theses and Dissertations
Identifier (type = local)
rucore19991600001
Location
PhysicalLocation (authority = marcorg); (displayLabel = Rutgers, The State University of New Jersey)
NjNbRU
Identifier (type = doi)
doi:10.7282/T3DV1K2R
Genre (authority = ExL-Esploro)
ETD doctoral
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Rights

RightsDeclaration (AUTHORITY = GS); (ID = rulibRdec0006)
The author owns the copyright to this work.
Copyright
Status
Copyright protected
Availability
Status
Open
RightsEvent (AUTHORITY = rulib); (ID = 1)
Type
Permission or license
Detail
Non-exclusive ETD license
AssociatedObject (AUTHORITY = rulib); (ID = 1)
Type
License
Name
Author Agreement License
Detail
I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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Technical

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ETD
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application/pdf
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application/x-tar
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7495680
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