Abstract
The expansion of a tandemly repeated trinucleotide sequence, CAG, is the mutational mechanism for several human genetic diseases. We present a generally applicable PCR amplification method using a fluorescently labelled locus specific primer flanking the CAG repeat together with paired primers amplifying from multiple priming sites within the CAG repeat. Triplet repeat primed PCR (TP PCR) gives a characteristic ladder on the fluorescence trace enabling the rapid identification of large pathogenetic CAG 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 alleles on Southern blot analysis were all correctly identified by TP PCR. This method is applicable for any human diseases involving CAG repeat expansions.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ashley C. T., Jr, Warren S. T. Trinucleotide repeat expansion and human disease. Annu Rev Genet. 1995;29:703–728. doi: 10.1146/annurev.ge.29.120195.003415. [DOI] [PubMed] [Google Scholar]
- Cheng S., Barceló J. M., Korneluk R. G. Characterization of large CTG repeat expansions in myotonic dystrophy alleles using PCR. Hum Mutat. 1996;7(4):304–310. doi: 10.1002/(SICI)1098-1004(1996)7:4<304::AID-HUMU3>3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
- Fu Y. H., Pizzuti A., Fenwick R. G., Jr, King J., Rajnarayan S., Dunne P. W., Dubel J., Nasser G. A., Ashizawa T., de Jong P. An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science. 1992 Mar 6;255(5049):1256–1258. doi: 10.1126/science.1546326. [DOI] [PubMed] [Google Scholar]
- Nagafuchi S., Yanagisawa H., Sato K., Shirayama T., Ohsaki E., Bundo M., Takeda T., Tadokoro K., Kondo I., Murayama N. Dentatorubral and pallidoluysian atrophy expansion of an unstable CAG trinucleotide on chromosome 12p. Nat Genet. 1994 Jan;6(1):14–18. doi: 10.1038/ng0194-14. [DOI] [PubMed] [Google Scholar]
- Neil D. L., Jeffreys A. J. Digital DNA typing at a second hypervariable locus by minisatellite variant repeat mapping. Hum Mol Genet. 1993 Aug;2(8):1129–1135. doi: 10.1093/hmg/2.8.1129. [DOI] [PubMed] [Google Scholar]
- Warner J. P., Barron L. H., Brock D. J. A new polymerase chain reaction (PCR) assay for the trinucleotide repeat that is unstable and expanded on Huntington's disease chromosomes. Mol Cell Probes. 1993 Jun;7(3):235–239. doi: 10.1006/mcpr.1993.1034. [DOI] [PubMed] [Google Scholar]