A rapid and accurate DHPLC assay for determination of apolipoprotein E genotypes.
The variants of apolipoprotein E (apoE) are closely related to hyperlipidemia III, Alzheimer's disease (AD), coronary artery disease (CAD) and many other human lipid metabolism-related problems. A rapid and accurate genotyping method specific for polymorphisms of the APOE gene is needed for population screening as well as diagnosis of apoE-related diseases in both the research and clinical setting. A polymerase chain reaction (PCR) method was designed to generate a 191-bp amplicon, which contains two common polymorphisms located in codons 112 and 158 of exon 4 of the APOE gene. The PCR amplicons for each sample were subjected to denaturing high-performance liquid chromatography (DHPLC) analysis, which was performed under partially denaturing conditions as determined by profiling the mixture of a tested sample and a homozygous standard control amplicon at the given ratio. A total of 297 DNA samples from Chinese population were enrolled to evaluate the specificity of the assay. A blinded validation study was then performed on 130 samples randomly selected from each of the six genotype groups as determined by DHPLC profiling. The genotypes obtained with the DHPLC method were in full agreement with those obtained by direct sequencing (130/130). We have developed a PCR/DHPLC genotyping assay capable of simultaneously determining all six genotypes of APOE gene in unknown test samples at one time.
Zeng
Yang
Y
Technology Center for Prenatal Diagnosis and DNA Testing of Genetic Disorders, Nanfang Hospital, Guangzhou 510515, Guangdong, P.R. China.
Miao
Fei
F
Li
Liang
L
Sun
De-Hua
DH
Xu
Xiang-Min
XM
D000544:Alzheimer Disease / Q000235:genetics; D001057:Apolipoproteins E / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003062:Codon / Q000235:genetics; D003324:Coronary Artery Disease / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005091:Exons / Q000235:genetics; D005838:Genotype; D006801:Humans; D006949:Hyperlipidemias / Q000235:genetics; D008403:Mass Screening; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction
D001057:Apolipoproteins E; D003062:Codon; D017931:DNA Primers