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Rapid and enhanced detection of mitochondrial DNA variation using single‐strand conformation analysis of superposed restriction enzyme fragments from polymerase chain reaction‐amplified products
Author(s) -
Barros Francisco,
Lareu María Victoria,
Salas Antonio,
Carracedo Angel
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150180110
Subject(s) - restriction enzyme , single strand conformation polymorphism , mitochondrial dna , dna , biology , microbiology and biotechnology , enzyme , polymerase chain reaction , silver stain , restriction fragment length polymorphism , genetics , chemistry , biochemistry , gene
Abstract A strategy is described for detecting mitochondrial (mt) DNA variation which permits rapid and straightforward screening for forensic purposes. The method is based on the selection of fragments with adequate length for performing single strand conformation polymorphism (SSCP) analysis selecting a set of restriction enzymes (RE) which yield fragments with prefixed lengths. After digestion of mtDNA by the appropriate enzyme or set of enzymes, SSCP analysis is performed in a semiautomatic electrophoretic system using a silver staining detection method. The conformational changes due to single mutations were therefore found not to change the electrophoretic protocol but to change the relative position of the mutations within the fragment. The discrimination power of this method is estimated to be 90% when two restriction enzymes ( Msp I and Hin fl) are used, but it is considerably higher when other enzymes are added.