HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Data Supplements Submit an electronic Letter to the Editor about this paper Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Smith, M. J. Articles by Fox, K. R. Search for Related Content PubMed PubMed Citation Articles by Smith, M. J. Articles by Fox, K. R. Related Collections Molecular Diagnostics and Genetics Lipids, Lipoproteins, and Cardiovascular Risk Factors

Combination of His-Tagged T4 Endonuclease VII with Microplate Array Diagonal Gel Electrophoresis for High-Throughput Mutation Scanning

¹ Human Genetics Division, School of Medicine, Southampton University Hospital, Southampton, UK;² Department of Physiology, Federal University of Para, Belem-Para, Brazil;³ School of Biological Sciences, University of Southampton, Southampton, UK;

^aaddress correspondence to this author at: Human Genetics Division, Duthie Building (Mp808), School of Medicine, Southampton University Hospital, Tremona Road, Southampton SO16 6YD, UK; fax 44-(0)23-80794264, e-mail inmd@soton.ac.uk

The first 300 words of the full text of this article appear below.

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.

Plasmid pRB210 (T4 endonuclease VII in pET11a) was a kind gift from Professor B. Kemper (Institute for Genetics, University of Cologne, Germany). The PCR primers used to amplify the endoVII gene from pRB210 were as follows: forward, 5'-GCGCCATATGATGTTATTGAC-3'; reverse, 5'-CAGCGGATCCTCATTTTAAACT-3'. After trimming was performed with BamHI and NdeI (New England Biolabs), pETendoVII was generated by ligation into pET15b (Novagen). . . . [Full Text of this Article]