Allele-specific associated polymorphism analysis: Novel modification of SSCP for mutation detection in heterozygous alleles using the paradigm of resistance to thyroid hormone

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Abstract
Allele‐specific polymorphism (ASAP) analysis is a modification of single‐strand conformation polymorphism (SSCP) mutation screening under optimized temperature conditions in a minigel format withethidium bromide detection. ASAP analysis was used to screen for and identify mutations within thehuman thryoid hormone receptor‐ß (hTR‐ß) gene. These mutations are the underlying cause ofresistance to thyroid hormone (RTH). Eleven dissimilar known hTR‐ß mutations and six previouslyuncharacterized mutations were accurately identified. ASAP screening extends to unique ASAP‐DNAfingerprinting as an identifying signature for each novel hTR‐ß mutation detected thus far. Gel‐plugsfrom the SSCP gels containing polymorphic single‐stranded DNA alleles were used without elution toprepare solid‐phase sequencing templates for mutant allele PCR and sequencing (MAPS). The couplingof ASAP analysis with MAPS has eliminated many of the interpretative and technical problemsassociated with the sequencing of heterozygous alleles. Together, this convenient screening and sequencing methodology offers accuracy, reproducibility, speed, and the potential elimination of allradioactivity, providing a general strategy for future automated detection and characterization of genetic mutations.

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