Abstract
Molecular genetic testing of myotonic dystrophy type 1 (DM1) is based on the identification and determination of a cytosine-thymine-guanine (CTG) repeat expansion in the DMPK gene. This is usually done by Southern blot analysis—a time-consuming and very laborious technique requiring high molecular weight DNA. The aim of our study was to develop a highly sensitive, rapid, and cost-effective molecular analysis characterizing the CTG repeat region of the DMPK gene based on a two-step polymerase chain reaction (PCR) protocol. (1) For the detection of alleles of up to 100 repeats, a quantitative fluorescent (QF) amplification with primers flanking the repeat region of the DM1 locus and two reference genes (PAX2 and DHCR7) for standardization was used. By this method it was possible to identify both homozygous and heterozygous DM1 alleles. (2) Long PCR was only performed if a single wild-type allele was detected that gave a QF-PCR signal of only half intensity compared to a homozygous sample. The results obtained using combined QF and Long PCR are highly accurate compared with Southern blot analysis. We conclude that our new rapid analysis is reliable for genetic testing of DM1 patients.