Application of FTA (R) sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR-based southern blotting

Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein ki nase gene. The clinical se verity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied o n by Southern blot analysis of restriction enzyme-digested genomic DNA. The development of PCR-based Southern blotting methodology provides a much mor e sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the us e of PCR on the diagnosis of DM. A modified PCR protocol to amplify differe nt lengths of CTG repeat region using various concentrations of 7-deaza-dGT P has been reported (1). Here we describe a procedure including sample coll ection, DNA purification, and PCR analysis of CTG repeat length without usi ng 7-deaza-dGTP. This protocol is very sensitive and convenient because onl y a small number of nucleate cells are needed for detection of CTG expansio n. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM. J. Clin. Lab. Anal. 13:188-193, 1999. ( C) 1999 Wiley-Liss, Inc.