Methylation-sensitive, single-strand conformation analysis (MS-SSCA): A rapid method to screen for and analyze methylation.
We have developed methylation-sensitive, single-strand conformation analysis (MS-SSCA) as a method of screening for methylation changes. Bisulfite modification converts cytosines to thymines, but methylated cytosines remain unchanged. This modification creates sequence differences between methylated and unmethylated samples, which can be resolved by SSCA. SSCA is 70-95% efficient at detecting single base changes in a fragment. As bisulfite modification of methylated DNA would typically involve several base changes in a fragment, the efficiency of detecting methylation using MS-SSCA could approach 100%. We applied this method to analyze the BRCA1 promoter CpG island in breast cancer samples. About 20% of sporadic breast cancers are hypermethylated at the BRCA1 promoter CpG island. MS-SSCA rapidly detected those tumors that had previously been shown to be methylated by Southern blotting. The variant bands detected by SSCA were analyzed by sequencing and shown to be methylated. MS-SSCA is a simple method for screening large numbers of samples for methylation and can accelerate genomic sequencing, as all bands can be isolated and sequenced directly.
Bianco
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Department of Haematology-Oncology and University of Adelaide Department of Medicine, Queen Elizabeth Hospital, Woodville, Australia.
Hussey
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Dobrovic
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D019313:BRCA1 Protein / Q000235:genetics; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D019175:DNA Methylation; D015226:Dinucleoside Phosphates / Q000235:genetics; D005260:Female; D019398:Genes, BRCA1; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D019313:BRCA1 Protein; D015226:Dinucleoside Phosphates; D007202:Indicators and Reagents; C015772:CpG dinucleotide