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Comprehensive, rapid and sensitive detection of sequence variants of human mitochondrial tRNA genes

Michikawa, Yuichi and Hofhaus, Götz and Lerman, Leonard S. and Attardi, Giuseppe (1997) Comprehensive, rapid and sensitive detection of sequence variants of human mitochondrial tRNA genes. Nucleic Acids Research, 25 (12). pp. 2455-2463. ISSN 0305-1048. http://resolver.caltech.edu/CaltechAUTHORS:MICnar97

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Abstract

In the present study, a comprehensive, rapid and sensitive method for screening sequence variation of the human mitochondrial tRNA genes has been developed. For this purpose, the denaturing gradient gel electrophoresis (DGGE) technique has been appropriately modified for simultaneous mutation analysis of a large number of samples and adapted so as to circumvent the problems caused by the anomalous electrophoretic behavior of DNA fragments encoding tRNA genes. Eighteen segments of mitochondrial DNA (mtDNA), each containing a single uniform melting domain, were selected to cover all tRNA-encoding regions using the computer program MELT94. All 18 segments were simultaneously analyzed by electrophoresis through a single broad range denaturing gradient gel under rigorously defined conditions, which prevent band broadening and other migration abnormalities from interfering with detection of sequence variants. All base substitutions tested, which include six natural mutations and 14 artificially introduced ones, have been detected successfully in the present study. Several types of evidence strongly suggest that the anomalous behavior in DGGE of tRNA gene-containing mtDNA fragments reflects their tendency to form temporary or stable alternative secondary structures under semi-denaturing conditions. The high sensitivity of the method, which can detect as low as 10% of mutant mtDNA visually, makes it valuable for the analysis of heteroplasmic mutations.


Item Type:Article
Additional Information:Copyright © 1997 by Oxford University Press Received January 31, 1997; Revised and Accepted April 24, 1997 These investigations were supported by NIH grant GM11726 and NIA grant AG12117-05 (to G.A.), NIH grant HG00345 (to L.L.) and by a Gosney Fellowship (to Y.M.). We are very grateful to Donald Johns, Ikuya Nonaka, Massimo Zeviani and Nathans Fischel-Ghodsian for making tissue samples and cell lines available to us and to David Page and Laura Brown for the samples of the wild-type and mutant human sry gene (HMG box DNA). We also thank Anne Chomyn and Antonio Enriquez for valuable discussions and Arger Drew, Benneta Keeley, Catherine Lin and Rosario Zedan for their expert technical assistance.
Record Number:CaltechAUTHORS:MICnar97
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:MICnar97
Alternative URL:http://nar.oxfordjournals.org/cgi/content/abstract/25/12/2455
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6431
Collection:CaltechAUTHORS
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Deposited On:08 Dec 2006
Last Modified:26 Dec 2012 09:21

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