A GENERAL-METHOD FOR THE DETECTION OF LARGE CAG REPEAT EXPANSIONS BY FLUORESCENT PCR

The expansion of a tandemly repeated trinucleotide sequence, CAG, is t he mutational mechanism for several human genetic diseases. We present a generally applicable PCR amplification method using a fluorescently labelled locus specific primer flanking the CAG repeat together with paired primers amplifying from multiple priming sites within the CAG r epeat. Triplet repeat primed PCR (TP PCR) gives a characteristic ladde r on the fluorescence trace enabling the rapid identification of large pathogenetic CAG repeats that cannot be amplified using flanking prim ers. We used our method to test a cohort of 183 people from myotonic d ystrophy families inducting unaffected subjects and spouses. Eighty fi ve clinically affected subjects with expanded alleles on Southern blot analysis were all correctly identified by TP PCR. This method is appl icable for any human diseases involving CAG repeat expansions.