TY - JOUR
T1 - Use of DNA toolbox for the characterization of mutation scanning methods. II
T2 - Evaluation of single-strand conformation polymorphism analysis
AU - Highsmith, W. Edward
AU - Nataraj, Arun J.
AU - Jin, Qian
AU - O'Connor, Jacquelyn M.
AU - El-Nabi, Sohbi H.
AU - Kusukawa, Noriko
AU - Garner, Mark M.
PY - 1999
Y1 - 1999
N2 - 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.
AB - 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.
KW - Mutation scanning
KW - Single-strand conformation polymorphism
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U2 - 10.1002/(SICI)1522-2683(19990101)20:6<1195::AID-ELPS1195>3.0.CO;2-A
DO - 10.1002/(SICI)1522-2683(19990101)20:6<1195::AID-ELPS1195>3.0.CO;2-A
M3 - Article
C2 - 10380759
AN - SCOPUS:0345279870
SN - 0173-0835
VL - 20
SP - 1195
EP - 1203
JO - ELECTROPHORESIS
JF - ELECTROPHORESIS
IS - 6
ER -