Development of a highly resolutive method, using a double quadruplex tetra-primer-ARMS-PCR coupled with capillary electrophoresis to study CD40LG polymorphisms. Issue 6 (December 2015)

• Record Type: Journal Article
• Authors: Aloui, Chaker; Sut, Caroline; Cognasse, Fabrice; Granados, Viviana; Hassine, Mohsen; Chakroun, Tahar; Garraud, Olivier; Laradi, Sandrine
• Is Part Of: Molecular and cellular probes. Volume 29:Issue 6(2015)
• Page Start: 335
• Page End: 342
• Publication Date: 2015-12
• Subjects: Quadruplex tetra-primer ARMS-PCR -- CD40LG -- SNP -- Inflammation -- Haplotype -- Population; Molecular probes -- Diagnostic use -- Periodicals; Pathology, Cellular -- Technique -- Periodicals; Cell Biology -- Periodicals; Molecular Biology -- Periodicals; Sondes moléculaires -- Utilisation diagnostique -- Périodiques; Cytopathologie -- Technique -- Périodiques; 572
• DOI: 10.1016/j.mcp.2015.11.003
• Languages: English
• ISSNs: 0890-8508

Abstract

Abstract: Polymorphisms in the CD40 ligand gene ( CD40LG ) are associated with various immunological disorders such as tumors, autoimmune and infectious diseases. The aim of this study was to develop a highly optimized double quadruplex tetra-primer amplification refractory mutation system PCR (double quadruplex T-ARMS-PCR) coupled with capillary electrophoresis to allow genotyping of eight relevant candidate CD40LG SNPs and to establish haplotypes. After conducting the double quadruplex T-ARMS-PCR, the genotypes obtained through agarose electrophoresis were compared with those obtained through capillary electrophoresis. This strategy was applied to analyze the genetic patterns of CD40LG in two distinct cohorts of blood donors (211 French and 274 Tunisian). The T-ARMS-PCR method was rapid, inexpensive, reproducible and reliable for SNP determination. Regarding the separation technique, capillary electrophoresis allows traceable and semi-automated analysis while agarose electrophoresis remains a cost-effective technique that does not require specialized or costly equipment. Using these methods, we identified significantly different genetic heterogeneity between the two investigated populations (p ≤ 0.0001) and we also extensively characterized their haplotypes. The obtained genotype distribution and the optimized quadruplex T-ARMS-PCR technique coupled with capillary electrophoresis provides valuable information for studying pathologic inflammation leading to various diseases in which CD40LG might be a candidate gene. Highlights: Double quadruplex T-ARMS-PCR provides valuable information to study diseases in which CD40LG might be a candidate gene. Capillary electrophoresis coupled with T-ARMS-PCR allows traceable, semi-automated analysis. This strategy was applied to analyze the genetic pattern of CD40LG in 2 distinct cohorts: French and Tunisian blood donors.