The objective of the proposed research are to contribute to the understanding and diagnosis of genetic human diseases by the development of a new technology based upon electrospray ionization mass spectrometry for rapid and accurate genotyping of single-nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Polymerase chain reaction (PCR) coupled with mass spectrometry is a near ideal platform for genotyping due to its high speed, accurate and precise mass measurements, and having the inherent ability to deduce primary structure via gas-phase sequencing. The proposed work will initially focus on elucidating the best strategy to prepare PCR products specifically for electrospray ionization mass spectrometry which is a formidable challenge. The precipitation or a more elaborate approach based on bio- affinity capture; both strategies can be automated in a 96-well format. Once a rapid preparation strategy is accomplished in the first 12-15 months of the proposed research, validation studies will be initiated gel- less, chip-less technology that is rapid, accurate and reliable for genotyping of SNPs and STRs which could realistically be placed in a clinical setting. Upon completion of the validation studies, unknown loci will be genotyped form the SNP panel of 90 to determine if there are any correlations between STR and SNP maps starting with the T-cell receptor beta loci located on chromosome 7. Concurrently, genes responsible for human deafness will be genotyped. All genotypes will be rapidly disseminated to the public through the development of a database including hyperlinks to other pertinent information regarding a specific polymorphic locus.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
3R01HG002159-02S2
Application #
6485517
Study Section
Genome Study Section (GNM)
Program Officer
Graham, Bettie
Project Start
2000-06-01
Project End
2002-05-31
Budget Start
2001-08-15
Budget End
2002-05-31
Support Year
2
Fiscal Year
2001
Total Cost
$66,950
Indirect Cost
Name
Virginia Commonwealth University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Frahm, J L; Muddiman, D C (2005) Nucleic Acid analysis by fourier transform ion cyclotron resonance mass spectrometry at the beginning of the twenty-first century. Curr Pharm Des 11:2593-613
Frahm, Jennifer L; Muddiman, David C; Burke, Michael J (2005) Leveling response factors in the electrospray ionization process using a heated capillary interface. J Am Soc Mass Spectrom 16:772-8
Nepomuceno, Angelito I; Mason, Christopher J; Muddiman, David C et al. (2004) Detection of genetic variants of transthyretin by liquid chromatography-dual electrospray ionization fourier-transform ion-cyclotron-resonance mass spectrometry. Clin Chem 50:1535-43
Hawkridge, Adam M; Zhou, Li; Lee, Milton L et al. (2004) Analytical performance of a venturi device integrated into an electrospray ionization fourier transform ion cyclotron resonance mass spectrometer for analysis of nucleic acids. Anal Chem 76:4118-22
Null, Allison P; Benson, Linda M; Muddiman, David C (2003) Enzymatic strategies for the characterization of nucleic acids by electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 17:2699-706
Benson, Linda M; Null, Allison P; Muddiman, David C (2003) Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses. J Am Soc Mass Spectrom 14:601-4
Null, Allison P; Nepomuceno, Angelito I; Muddiman, David C (2003) Implications of hydrophobicity and free energy of solvation for characterization of nucleic acids by electrospray ionization mass spectrometry. Anal Chem 75:1331-9
Nepomuceno, Angelito I; Muddiman, David C; Bergen 3rd, H Robert et al. (2003) Dual electrospray ionization source for confident generation of accurate mass tags using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry. Anal Chem 75:3411-8
Flora, Jason W; Null, Allison P; Muddiman, David C (2002) Dual-micro-ESI source for precise mass determination on a quadrupole time-of-flight mass spectrometer for genomic and proteomic applications. Anal Bioanal Chem 373:538-46
Flora, Jason W; Muddiman, David C (2002) Gas-phase ion unimolecular dissociation for rapid phosphopeptide mapping by IRMPD in a Penning ion trap: an energetically favored process. J Am Chem Soc 124:6546-7

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