A single-strand conformation polymorphism method for the large-scale analysis of mutations/polymorphisms using capillary array electrophoresis.
We present a high-throughput single-strand conformation polymorphism (SSCP) method, performed on a commercially available capillary array DNA sequencer. We tested various sieving matrices and electrophoretic conditions, using 51 DNA fragments which included 45 fragments carrying only one single nucleotide polymorphism (SNP), 4 fragments having two SNPs and 2 fragments with insertion or deletion. Resolution of alleles was improved by increasing concentrations of both sieving matrices and buffers, and all examined polymorphisms of DNA fragments were detected, most of them (45 fragments) as clearly split allele peaks in heterozygotes. Allele frequencies of SNPs can be estimated accurately by determining the relative amounts of alleles in pooled DNA. In this method, the turn-around time for the analysis of 96 samples is less than 3 h. These results demonstrate that capillary array-based SSCP is an efficient and accurate technique for the large-scale quantitative analysis of mutations/polymorphisms.
Kukita
Yoji
Y
Division of Genome Analysis, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
Higasa
Koichiro
K
Baba
Shingo
S
Nakamura
Michihiro
M
Manago
Sachi
S
Suzuki
Akari
A
Tahira
Tomoko
T
Hayashi
Kenshi
K
D004252:DNA Mutational Analysis / Q000295:instrumentation* / Q000592:standards; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods / Q000592:standards; D005787:Gene Frequency / Q000235:genetics; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity