Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO

Åkerman, Björn; Tuite, Eimer

doi:10.1093/nar/24.6.1080

Cited by 115 publications

(153 citation statements)

References 23 publications

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“…Of bottom-side detected DNA, 66 Ϯ 7% showed a corresponding fluorescence signal on the top side (n ϭ 8 chips). DNA molecules detected from the bottom but lacking top-side signal (red spots), were either due to polymerase stalling during the extension reaction or to possible release of the DNA strand after the first bottom-side imaging step (27). ZMWlocalized molecules without a bottom-side signal (green spots at ZMW locations) were attributed to aluminum surface, top-side attachments within the optical resolution of the ZMW location or ZMW side wall attachments.…”

Section: Resultsmentioning

confidence: 99%

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Korlach

Marks

Cicero

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

210

147

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Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of Ϸ30% were obtained for a range of ZMW diameters (70 -100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.fluorescence ͉ metal passivation ͉ microscopy ͉ polyvinyl phosphonic acid ͉ single molecule N anofabrication techniques have enabled new approaches to interrogate individual biomolecules by fluorescence techniques (reviewed in refs. 1 and 2). The extremely small size scale of the associated devices results in a drastic illumination volume reduction, allowing single-molecule investigations to take place at fluorophore concentrations increased to biologically relevant levels. In addition to higher temporal resolution and higher signal-to-noise ratios, they also provide spatial resolution beyond diffraction-limited optics.The zero-mode waveguide (ZMW) is one such nanophotonic confinement structure often consisting of a circular hole in a metal cladding film on a solid transparent substrate (3). In conjunction with laser-excited fluorescence, they provide observation volumes on the order of zeptoliters (10 Ϫ21 l), three to four orders of magnitude smaller than far-field excitation volumes. Applications of circular ZMWs have included the detection of single-molecule DNA polymerase activity by using labeled nucleotides at micromolar concentrations (3), the study of -repressor oligomerization dynamics (4), two-color crosscorrelation to rapidly screen for DNA restriction enzyme activity (5), and diffusion analysis of labeled membrane proteins in lipid bilayers of model membranes and living cells (6-11). C-shaped apertures have been described to study DNA hybridization interactions (12).ZMW technology applications have been limited by the unavailability of selective immobilization methods to position molecules exclusively in the observation volume, immediately above the transparent ZMW floor. One approach to selective immobilization exploits a feature inherent in the ZMW architecture. The transparent substrate and metal cladding are made of different materials, opening the possibility for a selective derivatization that will direct protein adsorption to the floor and not to the nearby metal walls. The nanometer size scale and three-dimensional n...

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Section: Resultsmentioning

confidence: 99%

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Korlach

Marks

Cicero

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

210

147

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“…These late cuts could either be due to random photoinduced cutting (16) or unspecific enzyme action. Because the photocutting rate is expected to have a square dependence on exposure time, we excluded cuts that occurred at 20 s or later from the further analysis.…”

Section: Resultsmentioning

confidence: 99%

Restriction mapping in nanofluidic devices

Riehn

Wang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

195

183

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We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100 -200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg 2؉ and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of Ϸ1.5 kbp in 1 min using single DNA molecules.R estriction mapping with endonucleases is a central method in molecular biology (1, 2). It is based on the measurement of fragment lengths after digestion, while possibly maintaining the respective order. We present here an approach to restriction mapping that is based on stretching DNA in nanofluidic channels, in which DNA is linearized to a length-independent fraction of its contour length (3, 4).To date, the most powerful method for constructing restriction maps of long DNA molecules (100 kbp and above) is the one developed by Schwartz and coworkers (5, 6). Their technique consists of stretching the DNA on a surface to establish a one-to-one mapping between spatial and genomic position, initiating the restriction by exposing the DNA to restriction enzymes and Mg 2ϩ , and optically observing the lengths of the resulting fragments. An elegant study of the same basic approach showing separation of specific binding and induced cutting was published by Taylor et al. (7).A fundamental principle in determining the error of a measurement, and a common strategy to reduce the experimental error, is to take multiple, statistically independent measurements of the same quantity. Note that the fixing of the molecule to the surface in Schwartz's approach prevents any fluctuations, and hence multiple molecules have to be observed to obtain statistically independent measurements of the same cut position. Moreover, genomic-length DNA molecules stretched on surfaces often exhibit inhomogeneous stretching, including breaks, and thus averaging over multiple molecules becomes imperative (6,8). In contrast, a molecule stretched inside a nanochannel is not subject to any forces other than those causing lateral confinement and thus is able to thermally relax and fluctuate around an equilibrium conformation. The evaluation of a single molecule is thus sufficient if complete digestion is achieved. A detailed description of the statistics and dynamics of DNA molecules in nanochannels is published in ref. 9.The main challenge in employing the concept of stretching and mapping DNA inside a closed fluidic system is to separate the steps of stretching and cutting. We have solved this problem by introducing the DNA electrophoretically into nanochannels and controlling the concentration of the enzyme cofactor Mg 2ϩ in the device shown in Fig. 1. It consists of a microfluidic ''loading'' channel containing the DNA to be analyzed, the restriction enzyme, and EDTA, and a microfluidic ''exit'' channel containing Mg 2ϩ and the restriction enzyme. The two microfluidic channels are linked by 10 n...

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“…Both methods that label polynucleotides prior to 31 delivering them to living cells, as well as "in situ" labeling methods will be discussed. 32 Polynucleotides have a well-defined primary and secondary structure, which enables yet 33 another classification of fluorophore interaction which is based on their sequence specificity. 34 Fluorophores that exhibit no sequence specificity interact with polynucleotides in a random 35 fashion and are spread all over the polynucleotide chain.…”

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confidence: 99%

“…25 The term "modified" stands for a nucleotide that differs from the naturally occurring 26 nucleotides through the addition of a functional moiety. At first, especially the nucleotides with 27 radioactive atoms ( 32 P, 33 P, 14 C, 3 H, 35 S) were used, but nowadays radioactive labels are often 28…”

mentioning

confidence: 99%

“…It should be noted that a 31 DNA or RNA primer which contains a modified nucleotide at the 3' end (generated using TdT 32 for example), can be used by DNA or RNA polymerases to be further elongated. Hence, 33 internally labeled polynucleotides can also be formed in this way. 5' EndTag nucleic acid 34 labeling kits are another available option from Vectorlabs, using T4 polynucleotide kinase to 35 transfer a thio-phosphate from ATPS to the 5' end of DNA, RNA or oligonucleotides.…”

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confidence: 99%

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Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

2015

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7Live-cell imaging has provided the life sciences with insights into the cell biology and 8 dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In 9 this review, we focus on the current fluorescent labeling strategies for nucleic acids, and in 10 particular messenger RNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad 11 range of scientific fields. By giving a background of the available techniques and an evaluation 12 of the pros and cons, we try to supply scientists with all the information needed to come to an 13 informed choice of nucleic acid labeling strategy aimed at their particular needs. 14

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Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO

Cited by 115 publications

References 23 publications

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Restriction mapping in nanofluidic devices

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

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