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Use of DNA toolbox for the characterization of mutation scanning methods. II: Evaluation of single‐strand conformation polymorphism analysis
Author(s) -
Highsmith W. Edward,
Nataraj Arun J.,
Jin Qian,
O'Connor Jacquelyn M.,
ElNabi Sohbi H.,
Kusukawa Noriko,
Garner Mark M.
Publication year - 1999
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/(sici)1522-2683(19990101)20:6<1195::aid-elps1195>3.0.co;2-a
Subject(s) - single strand conformation polymorphism , amplicon , dna , polymerase chain reaction , genetics , base pair , computational biology , microbiology and biotechnology , biology , gene
Single‐strand conformation polymorphism (SSCP) is one of the most commonly used methods for searching for unknown base changes (mutations). In order to characterize systematically the effects of important physical parameters on the sensitivity and specificity of SSCP, we used the DNA toolbox constructed as described in the companion paper [2]. Using this set of DNA molecules as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base, mutated to all other bases, were generated. The behavior of these constructs in manual and automated SSCP was analyzed as a function of the size, overall base content of the fragment, nature and location of the base change, and the temperature and pH of electrophoresis. Our results demonstrate that all of these variables interact to determine the rate of detection of single‐base changes, with the GC content being the predominant determinant of detection sensitivity.