Characterization of large CTG repeat expansions in myotonic dystrophy alleles using PCR

The CTG trinucleotide repeat expansions that are associated with myotonic dystrophy can be up to several thousand repeat units in length. We have developed a PCR protocol that has the potential to amplify mutant alleles with very large numbers of CTG repeats. The amplification uses the rTth DNA polymerase, XL system for long PCR targets together with primers which do not closely flank the repeat region and partial substitution of 7-deaza-dGTP for dGTP. Alleles containing up to approximately 800 CTG repeats were detected directly in agarose gels stained with ethidium bromide. Larger CTG repeat expansions required Southern blot transfer and detection with a repeat sequence probe; using this method, alleles containing up to approximately 2700 CTG repeats were detected. The PCR-based method described here was comparable to previous Southern blots of EcoRI-restriction digested genomic DNA in both the approximate size and heterogeneity of mutant alleles detected, but provided more precise sizes of the CTG repeat expansions than the restriction digest approach. This PCR protocol could potentially simplify current mutation detection protocols in the molecular diagnosis of myotonic dystrophy, and facilitate molecular studies of the disease. © 1996 Wiley-Liss, Inc.