In this proposal, we investigate a key component of the nanopore sequencing concept. Although the ultimate goal of the nanopore sequencing concept is to read the linear sequence of nucleotides without copying the DNA and without incorporating of labels, here, we explore of reading the linear sequence of a DNA by incorporating biotin labeled nucleotides. We plan to incorporate biotin labeled nucleotides to better differentiate the signal difference between all four nucleotides and conduct the basic research and technology development that are needed to read the linear sequence of a DNA. The strategy is to first increase the signal difference between bases in a DNA by incorporating Biotin-Labeled Nucleotides (BLN) into one of the four bases in the presence of the other natural nucleotides. For example, Biotin-Labeled Guanine (BLG) will be incorporated into DNA and then we will detect the locations of BLG by a single base resolution nanopore sensing system. We will investigate the ability to detect the positions of BLN in a single-stranded or double-stranded DNA molecule. One DNA molecule with all the same type of nucleotides substituted by a BLN, all G(xj) for example, will move at a controlled speed through a voltage biased solid state nanopore in an electrolyte solution. The translocating DNA will partially block the ionic current flow in the nanopore, and the current blockage signal generated will distinguish the difference between the biotin-labeled G(xj) and the rest of the bases as an electrical current signal GI(tj), here t is the time. Thus by calibrating the relation between xj and tj, the locations of G(x1), G(x2), ..., G(xj) can be estimated from GI(tj). The locations of the other three types nucleotides A(xj), T(xj), and C(xj) can be determined the same way. Thus, the whole sequence of a DNA molecule could be determined. Our initial goal is to sequence a DNA molecule of ~1000 base pair (bp) whose sequence is known. This will permit us to develop the basic technology necessary for solid-state nanopore-based sequencing. The specific goals in this proposal are: 1. Fabricate and test single base resolution (1-2 nm in thickness) solid-state nanopores. 2. Develop methods to control the rate of DNA translocation through a nanopore at ~100?s/base. 3. Optimize our nanopore sensing system and data analysis routines to determine the BLN(xj) in a ~1000 base long DNA. Study how accurate a high resolution solid-state nanopore device can determine the location of BLN(xj) in DNA. PROJECT

Public Health Relevance

In this proposal, we investigate a key component of the nanopore sequencing concept. Although the ultimate goal of the nanopore sequencing concept is to read the linear sequence of nucleotides without copying the DNA and without incorporating of labels, here, we explore of reading the linear sequence of a DNA by incorporating biotin labeled nucleotides. We plan to incorporate biotin labeled nucleotides to better differentiate the signal difference between all four nucleotides and conduct the basic research and technology development that are needed to read the linear sequence of a DNA. The strategy is to first increase the signal difference between bases in a DNA by incorporating Biotin-Labeled Nucleotides (BLN) into one of the four bases in the presence of the other natural nucleotides and then we will detect the locations of BLG by a single base resolution nanopore sensing system. We will investigate the ability to detect the positions of BLN in a singlestranded or double-stranded DNA molecule. Our initial goal is to sequence a DNA molecule of ~1000 base pair (bp) whose sequence is known. This will permit us to develop the basic technology necessary for solid-state nanopore-based sequencing.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Exploratory/Developmental Grants (R21)
Project #
3R21HG004776-03S1
Application #
8502006
Study Section
Special Emphasis Panel (ZHG1-HGR-N (M1))
Program Officer
Schloss, Jeffery
Project Start
2008-08-19
Project End
2013-11-30
Budget Start
2010-06-01
Budget End
2013-11-30
Support Year
3
Fiscal Year
2012
Total Cost
$181,310
Indirect Cost
$51,453
Name
University of Arkansas at Fayetteville
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
191429745
City
Fayetteville
State
AR
Country
United States
Zip Code
72701
Sugimoto, Manabu; Kato, Yuta; Ishida, Kentaro et al. (2015) DNA motion induced by electrokinetic flow near an Au coated nanopore surface as voltage controlled gate. Nanotechnology 26:065502
Rollings, Ryan; Graef, Edward; Walsh, Nathan et al. (2015) The effects of geometry and stability of solid-state nanopores on detecting single DNA molecules. Nanotechnology 26:044001
Hyun, Changbae; Kaur, Harpreet; McNabb, David S et al. (2015) Dielectrophoretic stretching of DNA tethered to a fiber tip. Nanotechnology 26:125501
Li, Jiali; Fologea, Daniel; Rollings, Ryan et al. (2014) Characterization of protein unfolding with solid-state nanopores. Protein Pept Lett 21:256-65
Hyun, Changbae; Kaur, Harpreet; Rollings, Ryan et al. (2013) Threading immobilized DNA molecules through a solid-state nanopore at >100 *s per base rate. ACS Nano 7:5892-900
Ando, Genki; Hyun, Changbae; Li, Jiali et al. (2012) Directly observing the motion of DNA molecules near solid-state nanopores. ACS Nano 6:10090-7
Rollings, Ryan C; McNabb, David S; Li, Jiali (2012) DNA characterization with ion beam-sculpted silicon nitride nanopores. Methods Mol Biol 870:79-97
Uplinger, James; Thomas, Brian; Rollings, Ryan et al. (2012) K(+) , Na(+) , and Mg(2+) on DNA translocation in silicon nitride nanopores. Electrophoresis 33:3448-57
Hyun, Changbae; Rollings, Ryan; Li, Jiali (2012) Probing Access Resistance of Solid-state Nanopores with a Scanning Probe Microscope Tip. Small 8:385-392
Hyun, Changbae; Rollings, Ryan; Li, Jiali (2012) Probing access resistance of solid-state nanopores with a scanning-probe microscope tip. Small 8:384-92

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