L Zhang1, Y Yang1, R Liu2, Q Li1, F Yang1, L Ma3, H Liu2, X Chen4, Z Yang5, L Cui6, Y He1. 1. Department of Cell Biology and Medical Genetics, Kunming Medical University, Kunming, Yunnan Province, China. 2. The First Affiliated Hospital, Kunming Medical University, Kunming, Yunnan Province, China. 3. Department of Histology and Embryology, Kunming Medical University, Kunming, Yunnan Province, China. 4. Kunming City Maternal and Child Health Hospital, Kunming, Yunnan Province, China. 5. Department of Pathogen Biology and Immunology, Kunming Medical University, Kunming, Yunnan Province, China. 6. Department of Entomology, The Pennsylvania State University, University Park, Pennsylvania, USA.
Abstract
INTRODUCTION: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect caused by G6PD gene mutations. This study aimed to develop a cost-effective, multiplex, genotyping method for detecting common mutations in the G6PD gene. METHODS: We used a SNaPshot approach to genotype multiple G6PD mutations that are common to human populations in South-East Asia. This assay is based on multiplex PCR coupled with primer extension reactions. Different G6PD gene mutations were determined by peak retention time and colors of the primer extension products. RESULTS: We designed PCR primers for multiplex amplification of the G6PD gene fragments and for primer extension reactions to genotype 11 G6PD mutations. DNA samples from a total of 120 unrelated G6PD-deficient individuals from the China-Myanmar border area were used to establish and validate this method. Direct sequencing of the PCR products demonstrated 100% concordance between the SNaPshot and the sequencing results. CONCLUSION: The SNaPshot method offers a specific and sensitive alternative for simultaneously interrogating multiple G6PD mutations.
INTRODUCTION:Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect caused by G6PD gene mutations. This study aimed to develop a cost-effective, multiplex, genotyping method for detecting common mutations in the G6PD gene. METHODS: We used a SNaPshot approach to genotype multiple G6PD mutations that are common to human populations in South-East Asia. This assay is based on multiplex PCR coupled with primer extension reactions. Different G6PD gene mutations were determined by peak retention time and colors of the primer extension products. RESULTS: We designed PCR primers for multiplex amplification of the G6PD gene fragments and for primer extension reactions to genotype 11 G6PD mutations. DNA samples from a total of 120 unrelated G6PD-deficient individuals from the China-Myanmar border area were used to establish and validate this method. Direct sequencing of the PCR products demonstrated 100% concordance between the SNaPshot and the sequencing results. CONCLUSION: The SNaPshot method offers a specific and sensitive alternative for simultaneously interrogating multiple G6PD mutations.
Authors: Stefania Bertoncini; Ruth Blanco-Rojo; Carlos Baeza; Eduardo Arroyo-Pardo; María Pilar Vaquero; Ana Maria López-Parra Journal: Genet Test Mol Biomarkers Date: 2011-01-03
Authors: N Laouini; A Bibi; H Ammar; K Kazdaghli; F Ouali; R Othmani; S Amdouni; S Haloui; C A Sahli; L Jouini; S Hadj Fredj; H Siala; N Ben Romdhane; N E Toumi; S Fattoum; T Messsaoud Journal: Mol Biol Rep Date: 2012-10-14 Impact factor: 2.316