A general method for the detection of large GAG repeat expansions by fluorescent PCR

Abstract

The expansion of a tandemly repeated tri-nucleotide sequence, GAG, is the muta-tional mechanism for several human genetic diseases. We present a generally applicable PCR amplification method using a fluorescently labelled locus specific primer flanking the GAG repeat together with paired primers amplifying from mul-tiple priming sites within the GAG repeat. Triplet repeat primed PCR (TP PCR) gives a characteristic ladder on the fluor-escence trace enabling the rapid iden-tification of large pathogenetic GAG repeats that cannot be amplified using flanking primers. We used our method to test a cohort of 183 people from myotonic dystrophy families including unaffected subjects and spouses. Eighty five clinically affected subjects with expanded allelcs on Southern blot analysis were all correctly identified by TP PCR. This method is ap-plicable for any human diseases involving GAG repeat expansions.