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Imran Hussain, Xin Gong, Vinod V Mootha; Triplet repeat primed PCR assay to genotype the CTG18.1 trinucleotide repeat polymorphism in TCF4. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1025.
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© ARVO (1962-2015); The Authors (2016-present)
To genotype the CTG18.1 trinucleotide polymorphism using a combination of short tandem repeat (STR) analysis and triplet repeat primed polymerase chain reaction (TP-PCR) assay and validate our genotyping approach with Southern blot analysis. Fuchs’ dystrophy is strongly associated with the intronic expanded CTG18.1 trinucleotide repeat polymorphism in TCF4. The TP-PCR assay was originally described as a general method for the detection of CAG repeat expansion in myotonic dystrophy.
The CTG18.1 polymorphism was genotyped using genomic DNA from over 515 subjects using a combination of STR analysis and TP-PCR assay. On samples where STR analysis detected only one allele or failed to detect any alleles, TP-PCR was performed to confirm the presence of an expanded allele(s). Our TP-PCR assay utilized P1, a fluorescent primer designed to a region upstream from the CTG18.1 allele. The companion reverse primer P4 on the complementary strand was comprised of 5 units of the CTG repeat and a 5′ tail to serve as an anchor for a second reverse primer P3, which prevents progressive shortening of the PCR products during subsequent cycles. The 5′ tail of primer P4 and the “common” flag primer P3 share no homology with human sequence. PCR was performed with an initial denaturation of 9 min at 95°C, followed by 10 cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 4 min, and then 30 cycles of 95°C for 45 s, 62°C for 45 s, and 72°C for 4 min with a 15 s extension at each cycle. The final extension step was 72°C for 10 min. The TP-PCR products were analyzed on the ABI 3730XL DNA analyzer. Southern blot analysis was performed on 10 DNA samples.
The TP-PCR assay was able to resolve zygosity of the CTG18.1 allele on samples where the STR analysis revealed only one allele or no alleles. Characteristic tracing patterns of the CTG repeat primed electropherograms allowed us to distinguish samples that were truly homozygous for a stable CTG18.1 allele from those that harbored an expanded CTG18.1 allele that was undetectable by STR analysis. TP-PCR was also able to confirm the presence of two expanded CTG18.1 alleles in samples where STR analysis failed to detect any allele. Southern blot analysis on 10 samples validated our genotyping results obtained by STR and TP-PCR assays.
The combination of STR and TP-PCR assays is a simple and efficient method to genotype the CTG18.1 polymorphism.
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