Determination of the Apo E Genotype Using LightCyclerTM Apo E Mutation Detection Kit
Nednapis Tirawanchai PhD*, Anchaleekorn Somkasetrin MSc*, Chanok Avirutakarn MSc**, Narong Bunyaratvej MD***
Affiliation : * Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Bangkok, Thailand ** Roche Diagnostic Thailand *** Department of Orthopedics, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
Apolipoprotein E (Apo E) is found to be genetically polymorphic. There are 3 common alleles designated as E2, E3 and E4. Polymorphism of the Apo E DNA is associated with the risk increase of many diseases such as dyslipidemia, cardiovascular diseases, Alzheimer’s diseases, etc. Hence, the interest in genotyping of the Apo E is now rising for the purpose of pre-diagnosis. The aim of this study was to character- ize the Apo E DNA polymorphism using LightCycler(cid:31)Apo E mutation detection kit (Roche) in the Thai normal healthy subjects. The genomic DNA was extracted from the blood of 133 normal healthy subjects using DNA extraction kit (Roche). Exon 4 of the Apo E gene was amplified by the extracted genomic DNA using the real-time PCR. The simultaneous analysis of the two polymorphic codons (Codons 112 and 158) in a single reaction was conducted by using the two reporter dyes with the different excitation and emission spectrum LightCycler-Red 640 (LC-Red 640) and LC-Red 705 followed by the color compensation software to correct the temperature-dependent crossover among the emission spectra of the dyes. The different genotypes were then determined by performing the melting curve analysis in the two different channels. The results showed that the allele frequencies of the Apo E2, E3 and E4 were 0.26, 0.63 and 0.11 respectively and the genotype frequencies of the E2/3, E2/4, E3/3,E3/4 and E4/4 were 47.37, 5.26, 32.33, 14.28 and 0.75% respectively. This study found that E2/3 was the most common genotype of the Apo E. In conclusion, the LightCycler (LC) allelic discrimination method to genotype the Apo E is rapid, simple, reliable and strongly recommended for being successful diagnostic tests in the future.
Keywords : Apolipoproteins E, DNA, Mutation, Polymorphism, Genetic, Reagent kits, Diagnostic
