Real-time PCR Detection of the Recessive Dystrophic Epidermolysis Bullosa-associated c.2470insG Mutation in Unrelated Mexican Families.

[dystrophic epidermolysis bullosa]

Recessive dystrophic epidermolysis bullosa (R-DEB ) is caused by mutations in the COL 7 A 1 gene . The most common mutation reported in Mexican families is the c .2470 insG mutation , normally detected by DNA sequencing . We report a faster and more economical high -throughput genotyping method to detect the c .2470 insG mutation using specific TaqMan probes in a real-time polymerase chain reaction (RT-PCR ) that facilitates genotype analysis with allelic discrimination plots . Our new method correctly genotyped 45 samples that had previously been sequenced as 41 wild-type homozygous (-/-), 1 heterozygous (-/G ) and three mutant homozygous (G /G ) (100 % specificity ). This new method allows high -throughput screening and furthermore is economical ($3 US /sample ), fast (2 h ), and sensitive as it requires only 20 ng input DNA . We used the new test to genotype 89 individuals from 32 unrelated Mexican families with R-DEB . The observed genotypic frequencies were 93 .3 % for the homozygous wild-type and 6 .7 % for the heterozygous genotype . The homozygous mutant genotype was not found . In conclusion , the allelic discrimination assay by RT-PCR is a sensitive , specific and effective high -throughput test for detecting the c .2470 insG mutation .

Diseases
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Diseases presenting "been sequenced as" symptom

dystrophic epidermolysis bullosa

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