The proposed experiments will develop a powerful method for polynucleotide (PN) separations, High Performance Affinity Chromatography (PN-HPAC). PN-HPAC may provide the means for rapid, clinical diagnosis for inherited genetic disease where the lesion on DNA is known. Molecular biology, and its applications to human health and well-being should also benefit from the development of PN-HPAC. The method involves attaching a defined DNA sequence to macroporous silica supports and preparing a chromatography column. The attached DNA will specifically hybridize by base pairing with complementary PN strands, even in the presence of other PN sequences. The specificity of PN hybridization should allow highly selective chromatography; by analogy, PN-HPAC represents a sort of """"""""reversed Southern hybridization"""""""". Calculations presented suggest that small columns (1 x 10 mm) with kinetics should allow rapid separations to be performed. Preparatively, the columns will be used to rapidly separate mRNA, plasmids, and phages from crude cellular extracts. Analytically, the columns should be useful both clinically and for research; model studies testing the selectivity of PN-HPAC will demonstrate the feasibility of these applications. Other experiments and the susceptibility of isolated M13 phage PN- HPAC in recombinant DNA experiments and transfection of E. coli will be established. These model studies should establish PN-HPAC as a valuable tool for molecular biology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043609-03
Application #
3302700
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1989-08-01
Project End
1992-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Tennessee Health Science Center
Department
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Jia, Yinshan; Jarrett, Harry W (2015) Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose. Anal Biochem 482:1-6
Nagore, Linda I; Jarrett, Harry W (2015) T(3): targeted proteomics of DNA-binding proteins. Anal Biochem 474:8-15
Jia, Yinshan; Nagore, Linda; Jarrett, Harry (2015) Southwestern Blotting Assay. Methods Mol Biol 1334:85-99
Jia, Yinshan; Larionov, Oleg; Jarrett, Harry W (2014) Coupling of deoxyribonucleic acid to solid supports using 3' terminal ribose incorporation. J Chromatogr A 1339:73-9
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Jarrett, Harry W (2012) Proteomic methodologies to study transcription factor function. Methods Mol Biol 786:315-34
Zhou, Yanwen; Jia, Yinshan; Jarrett, Harry W (2012) Asymmetric polymerase chain reaction provides alternatives for preparation of (GT)ýýý-tailed duplex DNA promoter for promoter trapping. Anal Biochem 427:133-8
Jiang, Daifeng; Mummidi, Srinivas; Ahuja, Sunil K et al. (2011) CCR5 promoter haplotype transcription complex characterization. J Health Care Poor Underserved 22:73-90
Jiang, Daifeng; Jia, Yinshan; Jarrett, Harry W (2011) Transcription factor proteomics: identification by a novel gel mobility shift-three-dimensional electrophoresis method coupled with southwestern blot and high-performance liquid chromatography-electrospray-mass spectrometry analysis. J Chromatogr A 1218:7003-15

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