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Combination of His-tagged T4 endonuclease VII with microplate array diagonal gel electrophoresis for high-throughput mutation scanning

Combination of His-tagged T4 endonuclease VII with microplate array diagonal gel electrophoresis for high-throughput mutation scanning
Combination of His-tagged T4 endonuclease VII with microplate array diagonal gel electrophoresis for high-throughput mutation scanning
Various physical mutation-scanning methods have been developed to avoid unnecessary resequencing of long stretches of DNA (1)(2)(3)(4)(5)(6). Protein-based mutation-scanning techniques include enzymatic digestion [reviewed in Ref. (7)], protein binding to a DNA duplex, and direct analyses of the in vivo or in vitro gene product. One such enzyme is T4 endonuclease VII (endoVII), the product of gene 49 of bacteriophage T4 (8). Radiolabel replacement with fluorescent tags has facilitated automated analysis (9). EndoVII recognizes heteroduplex structural distortions, nicking 2–6 bp 3' to the distortion, with efficiency dependent on sequence context (10) and mismatch type(11). Perfectly matched DNA undergoes some background digestion, which produces a highly reproducible pattern (12). Mutation detection sensitivity obtained with endoVII digestion was found to be similar to that for denaturing HPLC and direct sequencing (13).
Microplate array diagonal gel electrophoresis (MADGE) (14) provides an open-faced 96-well gel format for polyacrylamide gels. Recently, nondenaturing 192-, 384-, and 768-well formats of MADGE for high-throughput checking of PCR and post-PCR reactions (15) have been developed. We have combined, in proof-of-principle experiments, the mismatch digestion properties of endoVII with the high-throughput capabilities of MADGE and a newly developed denaturing MADGE format to create a simple mutation-scanning technique that can screen 1000 PCR samples during a single 35-min electrophoretic run.
non-u.s.gov't, electrophoresis, indicators and reagents, histidine, humans, hypercholesterolemia, chemistry, oligonucleotide array sequence analysis
1043-1046
Smith, Matt J.
6f32073d-9994-49b5-bfa9-a559a5f3a6cf
Pante-de-Sousa, Gabriella
937f9f0b-2c54-41c8-8366-49b2b6ca62c3
Alharbi, Khalid K.
f8267cbb-ad0a-4ad8-b483-ff896223dfda
Chen, Xiao-he
fd93b896-d8cc-4b0e-a811-c0e6a8ff8308
Day, Ian N.M.
b749b30a-1f4c-40eb-af0e-a50427388b39
Fox, Keith R.
9da5debc-4e45-473e-ab8c-550d1104659f
Smith, Matt J.
6f32073d-9994-49b5-bfa9-a559a5f3a6cf
Pante-de-Sousa, Gabriella
937f9f0b-2c54-41c8-8366-49b2b6ca62c3
Alharbi, Khalid K.
f8267cbb-ad0a-4ad8-b483-ff896223dfda
Chen, Xiao-he
fd93b896-d8cc-4b0e-a811-c0e6a8ff8308
Day, Ian N.M.
b749b30a-1f4c-40eb-af0e-a50427388b39
Fox, Keith R.
9da5debc-4e45-473e-ab8c-550d1104659f

Smith, Matt J., Pante-de-Sousa, Gabriella, Alharbi, Khalid K., Chen, Xiao-he, Day, Ian N.M. and Fox, Keith R. (2005) Combination of His-tagged T4 endonuclease VII with microplate array diagonal gel electrophoresis for high-throughput mutation scanning. Clinical Chemistry, 51 (6), 1043-1046. (doi:10.1373/clinchem.2004.046755).

Record type: Article

Abstract

Various physical mutation-scanning methods have been developed to avoid unnecessary resequencing of long stretches of DNA (1)(2)(3)(4)(5)(6). Protein-based mutation-scanning techniques include enzymatic digestion [reviewed in Ref. (7)], protein binding to a DNA duplex, and direct analyses of the in vivo or in vitro gene product. One such enzyme is T4 endonuclease VII (endoVII), the product of gene 49 of bacteriophage T4 (8). Radiolabel replacement with fluorescent tags has facilitated automated analysis (9). EndoVII recognizes heteroduplex structural distortions, nicking 2–6 bp 3' to the distortion, with efficiency dependent on sequence context (10) and mismatch type(11). Perfectly matched DNA undergoes some background digestion, which produces a highly reproducible pattern (12). Mutation detection sensitivity obtained with endoVII digestion was found to be similar to that for denaturing HPLC and direct sequencing (13).
Microplate array diagonal gel electrophoresis (MADGE) (14) provides an open-faced 96-well gel format for polyacrylamide gels. Recently, nondenaturing 192-, 384-, and 768-well formats of MADGE for high-throughput checking of PCR and post-PCR reactions (15) have been developed. We have combined, in proof-of-principle experiments, the mismatch digestion properties of endoVII with the high-throughput capabilities of MADGE and a newly developed denaturing MADGE format to create a simple mutation-scanning technique that can screen 1000 PCR samples during a single 35-min electrophoretic run.

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More information

Published date: 1 June 2005
Keywords: non-u.s.gov't, electrophoresis, indicators and reagents, histidine, humans, hypercholesterolemia, chemistry, oligonucleotide array sequence analysis

Identifiers

Local EPrints ID: 60246
URI: http://eprints.soton.ac.uk/id/eprint/60246
PURE UUID: 39f5b2bf-4be3-4c54-b986-b5ff424bd694
ORCID for Keith R. Fox: ORCID iD orcid.org/0000-0002-2925-7315

Catalogue record

Date deposited: 08 Sep 2008
Last modified: 16 Mar 2024 02:36

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Contributors

Author: Matt J. Smith
Author: Gabriella Pante-de-Sousa
Author: Khalid K. Alharbi
Author: Xiao-he Chen
Author: Ian N.M. Day
Author: Keith R. Fox ORCID iD

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