Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations

Abstract Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases.

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Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.10.3522 ◽

1988 ◽

Vol 85 (10) ◽

pp. 3522-3526 ◽

Cited By ~ 15

Author(s):

C. Lopez-Galindez ◽

J. A. Lopez ◽

J. A. Melero ◽

L. de la Fuente ◽

C. Martinez ◽

...

Keyword(s):

Influenza Virus ◽

Genetic Variability ◽

Point Mutations ◽

Rnase A ◽

Mismatch Cleavage ◽

Cleavage Method

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An Improved Method for the Preparation and TEM Examination of Sharp Pointed Tips used in APFIM and STM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100134107 ◽

1990 ◽

Vol 48 (2) ◽

pp. 102-103

Author(s):

E.A. Fischione ◽

P.E. Fischione ◽

J.J. Haugh ◽

M.G. Burke

Keyword(s):

Atom Probe ◽

Preparation Process ◽

Improved Method ◽

Ion Milling ◽

Polishing Process ◽

Probe Field ◽

Scanning Tunnelling ◽

Stm Tips ◽

Tunnelling Microscopy ◽

Ion Microscopy

A common requirement for both Atom Probe Field-Ion Microscopy (APFIM) and Scanning Tunnelling Microscopy (STM) is a sharp pointed tip for use as either the specimen (APFIM) or the probe (STM). Traditionally, tips have been prepared by either chemical or electropolishing techniques. Recently, ion-milling has been successfully employed in the production of APFIM tips [1]. Conventional electropolishing techniques are applicable to a wide variety of metals, but generally require careful manual adjustments during the polishing process and may also be time-consuming. In order to reduce the time and effort involved in the preparation process, a compact, self-contained polishing unit has been developed. This system is based upon the conventional two-stage electropolishing technique in which the specimen/tip blank is first locally thinned or “necked”, and subsequently electropolished until separation occurs.[2,3] The result of this process is the production of two APFIM or STM tips. A mechanized polishing unit that provides these functions while automatically maintaining alignment has been designed and developed.

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Immunoelectron microscopic localization of elastic tissue in archival material using an improved method

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161539 ◽

1990 ◽

Vol 48 (3) ◽

pp. 794-795

Author(s):

J. C. Fanning ◽

J. F. White ◽

R. Polewski ◽

E. G. Cleary

Keyword(s):

Normal Animal ◽

Animal Tissue ◽

Elastic Tissue ◽

Human Tissues ◽

Improved Method ◽

Tissue Samples ◽

Archival Material ◽

Immuno Electron Microscopy ◽

Arteries And Veins ◽

Biochemical Examination

Elastic tissue is an important component of the walls of arteries and veins, of skin, of the lungs and in lesser amounts, of many other tissues. It is responsible for the rubber-like properties of the arteries and for the normal texture of young skin. It undergoes changes in a number of important diseases such as atherosclerosis and emphysema and on exposure of skin to sunlight.We have recently described methods for the localizationof elastic tissue components in normal animal and human tissues. In the study of developing and diseased tissues it is often not possible to obtain samples which have been optimally prepared for immuno-electron microscopy. Sometimes there is also a need to examine retrospectively samples collected some years previously. We have therefore developed modifications to our published methods to allow examination of human and animal tissue samples obtained at surgery or during post mortem which have subsequently been: 1. stored frozen at -35° or -70°C for biochemical examination; 2.

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