Visualization of a Specific Sequence on a Single Large DNA Molecule Using Fluorescence Microscopy Based on a New DNA-Stretching Method

1999 ◽  
Vol 265 (1) ◽  
pp. 140-143 ◽  
Author(s):  
Hidehiro Oana ◽  
Masanori Ueda ◽  
Masao Washizu
Keyword(s):  
Fluorescence Microscopy ◽  
Dna Stretching ◽  
Specific Sequence ◽  
Dna Molecule

scholarly journals Determining Trap Compliances, Microsphere Size Variations, and Response Linearities in Single DNA Molecule Elasticity Measurements with Optical Tweezers

2021 ◽  
Vol 8 ◽  
Author(s):  
Youbin Mo ◽  
Mounir Fizari ◽  
Kristina Koharchik ◽  
Douglas E. Smith
Keyword(s):  
Optical Tweezers ◽  
Dna Elasticity ◽  
Dna Stretching ◽  
Dna Molecule ◽  
Scale Factors ◽  
Length Measurements ◽  
Microsphere Size ◽  
Force Range ◽  
Single Dna Molecule ◽  
Displacement Measurements

We previously introduced the use of DNA molecules for calibration of biophysical force and displacement measurements with optical tweezers. Force and length scale factors can be determined from measurements of DNA stretching. Trap compliance can be determined by fitting the data to a nonlinear DNA elasticity model, however, noise/drift/offsets in the measurement can affect the reliability of this determination. Here we demonstrate a more robust method that uses a linear approximation for DNA elasticity applied to high force range (25–45 pN) data. We show that this method can be used to assess how small variations in microsphere sizes affect DNA length measurements and demonstrate methods for correcting for these errors. We further show that these measurements can be used to check assumed linearities of system responses. Finally, we demonstrate methods combining microsphere imaging and DNA stretching to check the compliance and positioning of individual traps.

scholarly journals Single long DNA molecule analysis using fluorescence microscopy

2000 ◽  
Vol 44 (1) ◽  
pp. 247-248 ◽  
Author(s):  
N. Kaji ◽  
M. Ueda ◽  
Y. Baba
Keyword(s):  
Fluorescence Microscopy ◽  
Dna Molecule

Existence of DNA in yeast microbodies

1978 ◽  
Vol 36 (2) ◽  
pp. 232-233
Author(s):  
Masako Osumi ◽  
Misuzu Nagano ◽  
Hiroko Kazama
Keyword(s):  
Catalase Activity ◽  
Buoyant Density ◽  
Model System ◽  
Contour Length ◽  
Native State ◽  
Normal Alkane ◽  
Remarkable Increase ◽  
Dna Molecule ◽  
Mitochondrial Dnas

We have found that microbodies appeared profusely together with a remarkable increase in catalase activity in normal alkane-grown cells of hydrocarbon-utilizing Candida yeasts, and that the microbodies multiplied by division in these cells. These features of Candida yeasts seem to provide a useful model system for studies on the biogenesis of the microbody. Subsequently, we have succeeded in isolation of Candida microbodies in an apparently native state, as judged biochemically and morphologically. The presence of DNA in the purified microbody fraction thus obtained was proved by the diphenylamine method. DNA molecule of about 15 urn in contour length was released from an isolated microbody. The physicochemical analyses of the microbody DNA revealed that its buoyant density differed from nuclear and mitochondrial DNAs. All these results lead us to the possibility that there is a novel type of DNA in microbodies.

Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

1988 ◽  
Vol 46 ◽  
pp. 118-119
Author(s):  
K. Jacobson ◽  
A. Ishihara ◽  
B. Holifield ◽  
F. Zhang
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Extended Period ◽  
Dynamic Structure ◽  
Living Cells ◽  
Membrane Dynamics ◽  
Molecular Cell Biology ◽  
Image Averaging ◽  
Single Living Cells ◽  
Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

Electron microscopic assays of restriction endonuclease cutting, exonuclease digestion, and hybridization

1984 ◽  
Vol 42 ◽  
pp. 686-687
Author(s):  
Mark Hannibal ◽  
Jacob Varkey ◽  
Michael Beer
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Restriction Endonuclease ◽  
Double Helix ◽  
Test Material ◽  
Electron Microscopic ◽  
Exonuclease Iii ◽  
Dna Molecule ◽  
Linear Molecules ◽  
Exonuclease Digestion

Workman and Langmore have recently proposed a procedure for isolating particular chromatin fragments. The method requires restriction endonuclease cutting of the chromatin and a probe, their digestion with two exonucleases which leave complimentary single strand termini and low temperature hybridization of these. We here report simple electron microscopic monitoring of the four reactions involved.Our test material was ϕX-174 RF DNA which is cut once by restriction endonuclease Xho I. The conversion of circles to linear molecules was followed in Kleinschmidt spreads. Plate I shows a circular and a linear DNA molecule. The rate of cutting is shown in Figure 1.After completion of the endonuclease cutting, one portion of the DNA was treated with exonuclease III, an enzyme known to digest the 3' terminals of double helical DNA. Aliquots when examined in the electron microscope reveal a decreasing length of double helix and increasing bushes at the ends.

Scanning x-ray fluorescence microscopy and principal component analysis

1992 ◽  
Vol 50 (2) ◽  
pp. 1752-1753
Author(s):  
Brian Cross
Keyword(s):  
Principal Component Analysis ◽  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
High Speed ◽  
Image Acquisition ◽  
Principal Component ◽  
Fluorescence Microscope ◽  
Acquisition Rate ◽  
X Ray ◽  
Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

Electron Microscopy of Bacteriophage T7 Internal Head Proteins

1975 ◽  
Vol 33 ◽  
pp. 638-639
Author(s):  
P. Serwer
Keyword(s):  
Electron Microscopy ◽  
Bacteriophage T7 ◽  
Specimen Preparation ◽  
Dna Molecule ◽  
The Core ◽  
Internal Core ◽  
Phage T7 ◽  
T7 Phage ◽  
Preparation Techniques ◽  
Internal Head

The genome of bacteriophage T7 is a duplex DNA molecule packaged in a space whose volume has been measured to be 2.2 x the volume of the B form of T7 DNA. To help determine the mechanism for packaging this DNA, the configuration of proteins inside the phage head has been investigated by electron microscopy. A core which is roughly cylindrical in outline has been observed inside the head of phage T7 using three different specimen preparation techniques.When T7 phage are treated with glutaraldehyde, DNA is ejected from the head often revealing an internal core (dark arrows in Fig. 1). When both the core and tail are present in a particle, the core appears to be coaxial with the tail. Core-tail complexes sometimes dislodge from their normal location and appear attached to the outside of a phage head (light arrow in Fig. 1).

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