Fluorescent Marker for Direct Detection of Specific dsDNA Sequences

Dylla‐Spears, Rebecca; Townsend, Jacqueline E.; Sohn, Lydia L.; Jen‐Jacobson, Linda; Müller, Susan

doi:10.1021/ac9019895

Cited by 24 publications

(55 citation statements)

References 32 publications

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“…The EcoRI protein was expressed from a maltose-binding protein–EcoRI (MBP–EcoRI) fusion construct [7,13]. Details of generation of the fusion gene, expression and cleavage of the fusion protein are provided in the supporting information available with the online version of this paper [7].…”

Section: Methodsmentioning

confidence: 99%

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Insights into copper coordination in the EcoRI–DNA complex by ESR spectroscopy

Tan

Jen‐Jacobson

et al. 2014

Molecular Physics

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The EcoRI restriction endonuclease requires one divalent metal ion in each of two symmetrical and identical catalytic sites to catalyse double-strand DNA cleavage. Recently, we showed that Cu2+ binds outside the catalytic sites to a pair of new sites at H114 in each sub-unit, and inhibits Mg2+ -catalysed DNA cleavage. In order to provide more detailed structural information on this new metal ion binding site, we performed W-band (~94 GHz) and X-band (~9.5 GHz) electron spin resonance spectroscopic measurements on the EcoRI–DNA–(Cu2+ )2 complex. Cu2+ binding results in two distinct components with different gzz and Azz values. X-band electron spin echo envelope modulation results indicate that both components arise from a Cu2+ coordinated to histidine. This observation is further confirmed by the hyperfine sub-level correlation results. W-band electron nuclear double resonance spectra provide evidence for equatorial coordination of water molecules to the Cu2+ ions.

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Section: Methodsmentioning

confidence: 99%

“…Details of generation of the fusion gene, expression and cleavage of the fusion protein are provided in the supporting information available with the online version of this paper [7]. The EcoRI protein was isolated, purified, and characterised as described in [7,13]. …”

Section: Methodsmentioning

confidence: 99%

Insights into copper coordination in the EcoRI–DNA complex by ESR spectroscopy

Tan

Jen‐Jacobson

et al. 2014

Molecular Physics

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“…Sequence-specific recognition of double-stranded DNA (dsDNA) without needing denaturation into single strands is attractive and important in disease diagnosis and human gene therapy, because DNA in its natural state is double-stranded (Dylla-Spears et al, 2009;Rogers et al, 2005). Recently, numerous methods including electrochemistry (Miao et al, 2014;Zhu et al, 2010;Patterson et al, 2010), fluorescent Zhao et al, 2009;Wu et al, 2011;Xiao et al, 2013), dynamic light scattering and colorimetric (McKenzie et al, 2008) have been used for sequence-specific recognition of dsDNA.…”

Section: Introductionmentioning

confidence: 99%

Enhanced electrochemical recognition of double-stranded DNA by using hybridization chain reaction and positively charged gold nanoparticles

Miao

Xing

et al. 2015

Biosensors and Bioelectronics

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“…We demonstrate proof of principle using planar extensional flow in a cross slot to trap and extend single, site-specifically-tagged dsDNA molecules. Using this method, we identify the positions of five known EcoRI target sites on λ-DNA that are tagged with a previously characterized mutant EcoRI-based fluorescent marker 31 . We determine our method’s accuracy and precision and compare the detection resolution to other single-molecule methods.…”

Section: Introductionmentioning

confidence: 99%

Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point

et al. 2010

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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.

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Fluorescent Marker for Direct Detection of Specific dsDNA Sequences

Cited by 24 publications

References 32 publications

Insights into copper coordination in the EcoRI–DNA complex by ESR spectroscopy

Insights into copper coordination in the EcoRI–DNA complex by ESR spectroscopy

Enhanced electrochemical recognition of double-stranded DNA by using hybridization chain reaction and positively charged gold nanoparticles

Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point

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