Background: Human genome dynamic mutations are a new class of gene mutations represented by an unstable number of trinucleotide repeats and causing severe human hereditary neuromuscular and neurodegenerative diseases. The identification of pathological expanded alleles on the molecular level is important for clinical diagnostics.
Methods and results: For the molecular diagnostics of expanded tandem repeat trinucleotide sequences we have introduced a fast and efficient TP-PCR fluorescent method according to Warner et al. (1996). We have modified this TP-PCR method for a rapid detection of expanded CTG alleles of the DMPK gene (myotonic dystrophy, MD) into a two-level protocol; first, the heterozygote sample DNAs were selected using P1/P2 primers flanking repeat tracts and, second, the TP-PCR protocol used was focused above all on the identification of a pathological allele. A fluorescent-labelled specific primer in TP-PCR was used for the exact determination of the number of CAG repeats of the gene IT-15 (Huntington's disease--HD) in the diagnostically important region of the grey zone (35 to 39 CAG). The reproducibility of the PCR results was demonstrated on control DNA samples with the known genotype and, in the case of MD, also by Southern blot analysis. We have especially shown the possibility of a cheaper PCR-P1/P2 and TP-PCR protocol, which can be used, with silver staining of separated PCR products on polyacrylamide gels.
Conclusions: Our experience with introducing the above-mentioned PCR methods into laboratory practice clearly documents the possibilities of their general applicability in the molecular diagnostics of hereditary diseases characterised by instability of the trinucleotide repeat tracts.