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

María G. Moreno-Treviño, Rafael B.R. León-Cachón, Francisco González-Salazar, Marcelino Aguirre-Garza, Ricardo M. Cerda-Flores, Irene Meester, Julio C. Salas-Alanis

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Recessive dystrophic epidermolysis bullosa (R-DEB) is caused by mutations in the COL7A1 gene. The most common mutation reported in Mexican families is the c.2470insG mutation, normally detected by DNA sequencing. We report a faster and more economical high-throughput genotyping method to detect the c.2470insG 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.2470insG mutation.

Original languageEnglish
Pages (from-to)596-599
Number of pages4
JournalArchives of Medical Research
Volume45
Issue number7
DOIs
Publication statusPublished - 1 Jan 2014

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Epidermolysis Bullosa Dystrophica
Real-Time Polymerase Chain Reaction
Mutation
Genotype
High-Throughput Screening Assays
DNA Sequence Analysis
DNA
Genes

All Science Journal Classification (ASJC) codes

  • Medicine(all)

Cite this

@article{22d8b463a88248aa8cdb1aa26c232b89,
title = "Real-time PCR Detection of the Recessive Dystrophic Epidermolysis Bullosa-associated c.2470insG Mutation in Unrelated Mexican Families",
abstract = "Recessive dystrophic epidermolysis bullosa (R-DEB) is caused by mutations in the COL7A1 gene. The most common mutation reported in Mexican families is the c.2470insG mutation, normally detected by DNA sequencing. We report a faster and more economical high-throughput genotyping method to detect the c.2470insG 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.2470insG mutation.",
author = "Moreno-Trevi{\~n}o, {Mar{\'i}a G.} and Le{\'o}n-Cach{\'o}n, {Rafael B.R.} and Francisco Gonz{\'a}lez-Salazar and Marcelino Aguirre-Garza and Cerda-Flores, {Ricardo M.} and Irene Meester and Salas-Alanis, {Julio C.}",
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Real-time PCR Detection of the Recessive Dystrophic Epidermolysis Bullosa-associated c.2470insG Mutation in Unrelated Mexican Families. / Moreno-Treviño, María G.; León-Cachón, Rafael B.R.; González-Salazar, Francisco; Aguirre-Garza, Marcelino; Cerda-Flores, Ricardo M.; Meester, Irene; Salas-Alanis, Julio C.

In: Archives of Medical Research, Vol. 45, No. 7, 01.01.2014, p. 596-599.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Real-time PCR Detection of the Recessive Dystrophic Epidermolysis Bullosa-associated c.2470insG Mutation in Unrelated Mexican Families

AU - Moreno-Treviño, María G.

AU - León-Cachón, Rafael B.R.

AU - González-Salazar, Francisco

AU - Aguirre-Garza, Marcelino

AU - Cerda-Flores, Ricardo M.

AU - Meester, Irene

AU - Salas-Alanis, Julio C.

PY - 2014/1/1

Y1 - 2014/1/1

N2 - Recessive dystrophic epidermolysis bullosa (R-DEB) is caused by mutations in the COL7A1 gene. The most common mutation reported in Mexican families is the c.2470insG mutation, normally detected by DNA sequencing. We report a faster and more economical high-throughput genotyping method to detect the c.2470insG 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.2470insG mutation.

AB - Recessive dystrophic epidermolysis bullosa (R-DEB) is caused by mutations in the COL7A1 gene. The most common mutation reported in Mexican families is the c.2470insG mutation, normally detected by DNA sequencing. We report a faster and more economical high-throughput genotyping method to detect the c.2470insG 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.2470insG mutation.

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