Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics.
Mutation screening is widely used for molecular diagnostics of inherited disorders. Furthermore, it is anticipated that the present and future identification of genetic risk factors for complex disorders will increase the need for high-throughput mutation screening technologies. Capillary array electrophoresis (CAE) SSCP analysis is a low-cost, automated method with a high throughput and high reproducibility. Thus, the method fulfills many of the demands to be met for application in routine molecular diagnostics. However, the need for performing the electrophoresis at three temperatures between 18 degrees C and 35 degrees C for achievement of high sensitivity is a disadvantage of the method. Using a panel of 185 mutant samples, we have analyzed the effect of sample purification, sample medium and separation matrix on the sensitivity of CAE-SSCP analysis to optimize the method for molecular diagnostic use. We observed different effects from sample purification and sample medium at different electrophoresis temperatures, probably reflecting the complex interplay between sequence composition, electrophoresis conditions and sensitivity in SSCP analysis. The effect on assay sensitivity from three different polymers was tested using a single electrophoresis temperature of 27 degrees C. The data suggest that a sensitivity of 98-99% can be obtained using a 10% long chain poly-N,N-dimethylacrylamide polymer.
Jespersgaard
Cathrine
C
Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark.
Larsen
Lars Allan
LA
Baba
Shingo
S
Kukita
Yoji
Y
Tahira
Tomoko
T
Christiansen
Michael
M
Vuust
Jens
J
Hayashi
Kenshi
K
Andersen
Paal Skytt
PS
D000178:Acrylamides / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000592:standards*; D019075:Electrophoresis, Capillary / Q000592:standards*; D030342:Genetic Diseases, Inborn / Q000175:diagnosis*; D005820:Genetic Testing; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000592:standards*; D018807:Polymorphism, Single-Stranded Conformational; D013696:Temperature
D000178:Acrylamides; C429790:poly(N,N-dimethylacrylamide)