Improved polymerase chain reaction conditions for quick diagnostics of Huntington disease.
Huntington disease (HD) belongs to a growing list of neurodegenerative disorders (fragile X syndrome [6], myotonic dystrophy [1], spino-bulbar muscular atrophy [2] etc.) characterized by unstable expanded trinucleotide repeats (so-called 'dynamic mutations'). The dynamic mutation causing HD represents the expansion of CAG triplets in the first exon of a gene IT15 (chromosome 4) coding for huntington. This trinucleotide stretch is varying in the range of 11-34 in normal chromosomes and 39-121 in HD chromosomes. The most direct diagnostic approach is to amplify the proximal region of IT15 gene (from patients genomic DNA) by polymerase chain reaction (PCR) and estimate the number of CAG triplets. All protocols published to date are difficult to reproduce because amplification is inefficient giving additional non-specific products. The strategy of our experiment is shown in Fig. 1. We designed one new primer, primer No. 2 (another primer was primer No. 1) and novel PCR conditions. Primer No. 2 is located closer to CAG triplets and its extension is not including the GC rich region. PCR amplified products, using primer Nos. 1 and 2, thus do not include the GC rich region and, therefore, are much more efficiently amplified (compared to the products of amplification with primer Nos. 1 and 3).
Culjković
B
B
Institute for Biological Research, Belgrade, Yugoslavia.
Ruzdijić
S
S
Rakić
L
L
Romac
S
S
D002874:Chromosome Mapping; D002894:Chromosomes, Human, Pair 4; D017931:DNA Primers; D005091:Exons; D006801:Humans; D006816:Huntington Disease / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D008214:Lymphocytes; D016133:Polymerase Chain Reaction / Q000379:methods*; D018911:Trinucleotide Repeats
D017931:DNA Primers