The urgent need to develop low-cost and high-quality revolutionary technologies for sequencing mammalian-sized genomes has inspired many experimental strategies. Chief among these is the nanopore-based electrophoresis. While excellent progress is continuously being made with this technique, there are many challenges in reaching the goals of very high quality sequencing and fabricating massively parallel sequencing devices. These challenges stem from the physics of nanopore-based electrophoresis of DNA which needs to be understood from a fundamental scientific point of view. The proposed research deals with fundamental understanding of the behavior of DNA in nanopore environments under the influence of electric and hydrodynamic forces, and ratcheting forces from enzymes. We will investigate the challenges underlying several key system components in the goal of reducing the cost, increasing the speed, and increasing the accuracy of sequencing mammalian-sized genomes. The major challenges deal with slowing down DNA through nanopore, effects of specific ions, conformational fluctuations of DNA, effects of flow fields arising from hydrodynamics, salt concentration gradients, and electroosmotic flow, and fluctuations in the processivity of enzymes. We will use a combination of concepts from polymer physics, statistical mechanics theory, computer simulations, and numerical computation of coupled nonlinear equations to address polyelectrolyte statistics and dynamics, electrostatics, and hydrodynamics in the phenomena of DNA translocation. The proposed research, while being generally relevant to all nanopore-based experiments, will be hinged specifically on: (a) slowing down DNA and fundamental understanding of translocation, mediated by voltage, temperature, identity and amount of electrolyte, salt concentration gradient, and patterns on pore surface, (b) controlling the stochasticity in enzyme-ratcheted translocation and fundamental understanding of coupling among fluctuations in enzyme processivity, DNA conformational fluctuations, and electrophoretic drift-diffusion, and (c) designing optimum configuration of compact arrays of thousands of nanopores for massively parallel DNA sequencing without crosstalk between the units.

Public Health Relevance

Availability of low-cost technologies for DNA sequencing with very high quality is vital in identifying the origins of diseases and maintenance of public health. The proposed research addresses the challenges in fundamental understanding of several key system components in the development of low-cost and high-quality genome sequencing technologies.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG002776-11
Application #
8728977
Study Section
Special Emphasis Panel (ZHG1-HGR-N (M1))
Program Officer
Schloss, Jeffery
Project Start
2003-06-06
Project End
2017-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
11
Fiscal Year
2014
Total Cost
$262,876
Indirect Cost
$91,376
Name
University of Massachusetts Amherst
Department
Engineering (All Types)
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Singh, Sunil P; Muthukumar, M (2014) Electrophoretic mobilities of counterions and a polymer in cylindrical pores. J Chem Phys 141:114901
Larkin, Joseph; Henley, Robert Y; Muthukumar, Murugappan et al. (2014) High-bandwidth protein analysis using solid-state nanopores. Biophys J 106:696-704
Jeon, Byoung-jin; Muthukumar, Murugappan (2014) Polymer capture by ?-hemolysin pore upon salt concentration gradient. J Chem Phys 140:015101
Muthukumar, M (2014) Communication: Charge, diffusion, and mobility of proteins through nanopores. J Chem Phys 141:081104
Katkar, H H; Muthukumar, M (2014) Effect of charge patterns along a solid-state nanopore on polyelectrolyte translocation. J Chem Phys 140:135102
Muthukumar, M (2014) Macromolecular mechanisms of protein translocation. Protein Pept Lett 21:209-16
Mirigian, Stephen; Muthukumar, Murugappan (2013) Kinetics of particle wrapping by a vesicle. J Chem Phys 139:044908
Muthukumar, M; Nossal, Ralph (2013) Micellization model for the polymerization of clathrin baskets. J Chem Phys 139:121928
Anderson, Brett N; Muthukumar, Murugappan; Meller, Amit (2013) pH tuning of DNA translocation time through organically functionalized nanopores. ACS Nano 7:1408-14
Mahalik, J P; Hildebrandt, B; Muthukumar, M (2013) Langevin dynamics simulation of DNA ejection from a phage. J Biol Phys 39:229-45

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