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1.  Polymer-Monovalent Salt-Induced DNA Compaction Studied via Single-molecule Microfluidic Trapping 
Lab on a Chip  2011;12(3):647-651.
Polymer-monovalent salt-induced single-molecule DNA compaction/condensation in a microfluidic stagnation point flow was studied by analyzing both DNA compaction images and time trajectories. For the whole DNA compaction process we observed three successive steps: Step I, a relaxation process of the stretched DNA that occurs slowly along the whole DNA chain, Step II, nucleus formation and growth, and Step III, corresponding to a rapid compaction of the chain. A memory effect was observed between Steps I and III, and a new (intruder-induced) nucleation mode was observed for the first time. This study extends the use of the microfluidic stagnation point flow, which we have previously used for sequence detection and to measure enzyme kinetics site-specifically.
PMCID: PMC3322635  PMID: 22173785
2.  Labeling DNA for Single-Molecule Experiments: Methods of Labeling Internal Specific Sequences on Double-Stranded DNA 
Nanoscale  2011;3(8):3027-3039.
This review is a practical guide for experimentalists interested in specifically labeling internal sequences on double-stranded (ds) DNA molecules for single-molecule experiments. We describe six labeling approaches demonstrated in a single-molecule context and discuss the merits and drawbacks of each approach with particular attention to the amount of specialized training and reagents required. By evaluating each approach according to criteria relevant to single-molecule experiments, including labeling yield and compatibility with cofactors such as Mg2+, we provide a simple reference for selecting a labeling method for given experimental constraints. Intended for non-specialists seeking accessible solutions to DNA labeling challenges, the approaches outlined emphasize simplicity, robustness, suitability for use by non-biologists, and utility in diverse single-molecule experiments.
PMCID: PMC3322637  PMID: 21734993
3.  Peptide Nucleic Acids as Tools for Single-Molecule Sequence Detection and Manipulation 
Nano Letters  2010;10(11):4697-4701.
The ability to strongly and sequence-specifically attach modifications such as fluorophores and haptens to individual double-stranded (ds) DNA molecules is critical to a variety of single-molecule experiments. We propose using modified peptide nucleic acids (PNAs) for this purpose and implement them in two model single-molecule experiments where individual DNA molecules are manipulated via microfluidic flow and optical tweezers, respectively. We demonstrate that PNAs are versatile and robust sequence-specific tethers.
PMCID: PMC3322611  PMID: 20923183
DNA; Peptide Nucleic Acid; Optical Tweezer; Microfluidic Cross-Slot; Single-Molecule; Sequence-Specific
4.  Exploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping 
Lab on a Chip  2010;11(3):435-442.
We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing the cofactor magnesium, we determine the binding location of the enzyme by the cleavage process of dsDNA as in optical restriction mapping, however here the DNA need not be immobilized on a surface. We note that no special labeling of the enzyme is required, which makes it simpler than our previous scheme using stagnation point flows for sequence detection. Our accuracy in determining the location of the recognition site is comparable to or better than other single molecule techniques due to the fidelity with which we can control the linearization of the DNA molecules. In addition, since the cleavage process can be followed in real time, information about the cleavage kinetics, and subtle differences in binding and cleavage frequencies among the recognition sites, may also be obtained. Data for the five recognition sites for the type II restriction endonuclease EcoRI on λ-DNA are presented as a model system. While the roles of the varying fluid velocity and tension along the chain backbone on the measured kinetics remain to be determined, we believe this new method holds promise for a broad range of studies of DNA-protein interactions, including the kinetics of other DNA cleavage processes, the dissociation of a restriction enzyme from the cleaved substrate, and other macromolecular cleavage processes.
PMCID: PMC3322636  PMID: 21072428
5.  Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point 
Lab on a Chip  2010;10(12):1543-1549.
We demonstrate the use of a microfluidic stagnation point flow to trap and extend single molecules of double-stranded (ds) genomic DNA for detection of target sequences along the DNA backbone. Mutant EcoRI-based fluorescent markers are bound sequence-specifically to fluorescently labeled ds λ-DNA. The marker-DNA complexes are introduced into a microfluidic cross slot consisting of flow channels that intersect at ninety degrees. Buffered solution containing the marker-DNA complexes flows in one channel of the cross slot, pure buffer flows in the opposing channel at the same flow rate, and fluid exits the two channels at ninety degrees from the inlet channels. This creates a stagnation point at the center of a planar extensional flow, where marker-DNA complexes may be trapped and elongated along the outflow axis. The degree of elongation can be controlled using the flow strength (i.e., a non-dimensional flow rate) in the device. Both the DNA backbone and the markers bound along the stretched DNA are observed directly using fluorescence microscopy and the location of the markers along the DNA backbone is measured. We find that our method permits detection of each of the five expected target site positions to within 1.5 kb with standard deviations of <1.5 kb. We compare the method’s precision and accuracy at molecular extensions of 68% and 88% of the contour length to binding distributions from similar data obtained via molecular combing. We also provide evidence that increased mixing of the sample during binding of the marker to the DNA improves binding to internal target sequences of dsDNA, presumably by extending the DNA and making the internal binding sites more accessible.
PMCID: PMC3322643  PMID: 20358051
6.  Fluorescent marker for direct detection of specific dsDNA sequences 
Analytical chemistry  2009;81(24):10049-10054.
We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzyme’s binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker’s ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods.
PMCID: PMC2811260  PMID: 19908852
7.  Ex Vivo Sputum Analysis Reveals Impairment of Protease-dependent Mucus Degradation by Plasma Proteins in Acute Asthma 
Rationale: Airway mucus plugs, composed of mucin glycoproteins mixed with plasma proteins, are an important cause of airway obstruction in acute severe asthma, and they are poorly treated with current therapies.
Objectives: To investigate mechanisms of airway mucus clearance in health and in acute severe asthma.
Methods: We collected airway mucus from patients with asthma and nonasthmatic control subjects, using sputum induction or tracheal aspiration. We used rheological methods complemented by centrifugation-based mucin size profiling and immunoblotting to characterize the physical properties of the mucus gel, the size profiles of mucins, and the degradation products of albumin in airway mucus.
Measurements and Main Results: Repeated ex vivo measures of size and entanglement of mucin polymers in airway mucus from nonasthmatic control subjects showed that the mucus gel is normally degraded by proteases and that albumin inhibits this degradation. In airway mucus collected from patients with asthma at various time points during acute asthma exacerbation, protease-driven mucus degradation was inhibited at the height of exacerbation but was restored during recovery. In immunoblots of human serum albumin digested by neutrophil elastase and in immunoblots of airway mucus, we found that albumin was a substrate of neutrophil elastase and that products of albumin degradation were abundant in airway mucus during acute asthma exacerbation.
Conclusions: Rheological methods complemented by centrifugation-based mucin size profiling of airway mucins in health and acute asthma reveal that mucin degradation is inhibited in acute asthma, and that an excess of plasma proteins present in acute asthma inhibits the degradation of mucins in a protease-dependent manner. These findings identify a novel mechanism whereby plasma exudation may impair airway mucus clearance.
PMCID: PMC2724713  PMID: 19423716
airway mucus; rheology; neutrophil elastase; plasma; asthma exacerbation

Results 1-7 (7)