ALLELE-SPECIFIC ASSOCIATED POLYMORPHISM ANALYSIS - NOVEL MODIFICATIONOF SSCP FOR MUTATION DETECTION IN HETEROZYGOUS ALLELES USING THE PARADIGM OF RESISTANCE TO THYROID-HORMONE

Allele specific polymorphism (ASAP) analysis is a modification of sing le strand conformation polymorphism (SSCP) mutation screening under op timized temperature conditions in a minigel format with ethidium bromi de detection. ASAP analysis was used to screen for and identify mutati ons within the human thryoid hormone receptor-beta (hTR-beta) gene, Th ese mutations are the underlying cause of resistance to thyroid hormon e (RTH). Eleven dissimilar known hTR-beta mutations and six previously uncharacterized mutations were accurately identified, ASAP screening extends to unique ASAP-DNA fingerprinting as an identifying signature for each novel hTR-beta mutation detected thus far. Gel plugs from the SSCP gels containing polymorphic single stranded DNA alleles were use d without elution to prepare solid-phase sequencing templates for muta nt allele PCR and sequencing (MAPS). The coupling of ASAP analysis wit h MAPS has eliminated many of the interpretative and technical problem s associated with the sequencing of heterozygous alleles. Together, th is convenient screening and sequencing methodology offers accuracy, re producibility, speed, and the potential elimination of all radioactivi ty, providing a general strategy for future automated detection and ch aracterization of genetic mutations. (C) 1995 Wiley Liss, Inc.