TitleAnalysis of single nucleotide polymorphisms using molecular affinity separation and mass spectrometry
NameMisra, Ashish (author), Kim, Sobin (chair), Roth, Charles (internal member), Pedersen, Henrik (internal member), Chiew, Yee (internal member), Lee, KiBum (outside member), Rutgers University, Graduate School - New Brunswick,
SubjectChemical and Biochemical Engineering,
DescriptionSingle 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.
NoteIncludes bibliographical references (p. 73-78)
Noteby Ashish Misra
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.