Rapid and Direct Detection of Apolipoprotein E Genotypes Using Whole Blood from Humans

Abstract

Polymerase chain reaction (PCR) is a powerful molecular biological tool in the field of toxicity testing and diagnostics. The use of PCR for large-scale genetic testing requires an effective method of sample processing. Unfortunately, isolation of PCR-quality DNA is time-consuming. PCR performed directly on whole blood is preferred because of time efficiency, cost of the procedure, and possible automation for large-scale toxicity evaluation and diagnosis. The apolipoprotein E (APOE) gene contains two single-nucleotide polymorphisms (SNP) located at codons 112 and 158, producing three APOE protein isoforms known to be associated with the risks of developing cardiovascular disease and susceptibility to Alzheimer's disease. In the present study, an attempt was made to use the AnyDirect solution for APOE genotyping by PCR using whole blood directly without DNA purification. Results for two PCR methods, (1) conventional PCR using purified DNA and conventional buffer and (2) direct PCR using whole blood and AnyDirect solution, were compared in four different PCR-based APOE genotyping methods including PCR restriction-fragment-length polymorphism (PCR-RFLP), allele-specific PCR, SNaPshot mini-sequencing, and multiplex tetra-primer amplification refractory mutation system (T-ARMS) PCR. There was complete concordance in the APOE genotypes between conventional PCR and direct PCR, in all four different PCR-based APOE genotyping methods. Data demonstrated that the four different PCR-based APOE genotyping methods are able to determine the APOE genotypes successfully using whole blood directly with the use of AnyDirect solution. The direct multiplex T-ARMS PCR using whole blood may be the most rapid, simple, and inexpensive method for detecting APOE genotypes among four different APOE genotyping methods.

Selenium Level and Dyslipidemia in Rural Elderly Chinese

“…Genomic DNA was extracted with Bio Teke Whole Blood DNA Extraction Kits following the manufacturer’s instructions. Two single-nucleotide polymorphisms (SNPs) at triplet 112 (rs429385) and 158 (rs7412) were determined by the multiplex tetra-primer amplification refractory mutation system (T-ARMS) PCR reactions according to literature [ 28 ]. Participants were classified into two groups, APOEε 4 carriers ( ε 2/ ε 4, ε3 / ε 4, ε 4/ ε 4) and non-carriers ( ε 2/ ε 2, ε3 / ε 3, ε 2/ ε 3) in the analysis.…”

Human Plasma Lipid Modulation in Schistosomiasis Mansoni Depends on Apolipoprotein E Polymorphism

“…Single nucleotide APOE polymorphisms (rs7412 and rs429358) were detected by polymerase chain reaction (PCR) [31]. Amplified sequences were digested with the enzyme Hha I (5 units/mL) for 3 h and the restriction fragments were separated by 4% agarose gel electrophoresis and stained with ethidium bromide (0.5 mg/L).…”

Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

“…39,40 ApoE genotype was determined by restriction fragment length polymorphism analysis on polyacrylamide gel as previously described with slight modification. 41 Cells were grown in Eagle's minimal essential Medium (MEM; Invitrogen, Chicago, IL, USA) with 10% fetal bovine serum (FBS; Invitrogen) and with 1× penicillin/streptomycin (Pen/Strep; Invitrogen) at 37°C in a humidified environment containing 5% CO 2 . The identity of RPE cells from three donors was confirmed by cytokeratin-18 and ZO-1 stain (not shown).…”