RESUMO
Objective To establish a high speed and effective real-time PCR assay to analyze apolipoprotein E(apoE)genotyping in Chinese population using hybridization probes and melting curves. Methods Lightcycler was used to analyzed two codons'polymorphism after condition was optimized. The persons elder than 60 years including 133 patients with abdominal fat and 108 healthy elder were selected. The detection probes were labeled with L-Cred 640 and LC-Red 705 at 5'end covers codons 112 and 158 with the corresponding anchor probes labeled with fluorescein at 3'ends.A 265-bp fragment of the apoE gene was amplified from human genomic DNA to produce FRET.Depending on the various types of base-pair mismatch in the heteroduplex,wild type and mutant type were differentiated.Results The peaks represented the sequence-specific melting points(Tm) and each genetype showed perfect peak.E2/3 and E3/4 in abdominal fat group were much more common allele than health persons(x2=4.210.P＜0.004,x2=6·328,P＜0.012).The frequencies of abdominal fat group was E2/3(27.8%),E3/4(24.8%),E3/3(42.1%),E2/4(2.3%),E4/4(2.2%)and E2/2(0.8%).The frequencies of healthy controls were E2/3(16.7%),E3/4(12%),E3/3(68.5%),E2/4(1.9%),E4/4(O%)and E2/2(0.9%).It showed high agreement as compared with DNA sequencing analysis The expression of apoE in abdominal fat group (101.5±73.6)was up-regulated than the healthy group(50.6±27.1,P＜0.01).Conclusions Apolipoprotein E genotyping method by melting curve is faster and simpler than other technique. It can prevent the cross-contaminated and is suitable to be applied in clinical diagnosis.There was significant difference between the two groups.There was positive relationship between the elder's abdominal fat and apoE gene polymorphism. The genotyping of E3/4.E2/3 or E4/4 had the important role in the elder's abdominal fat on genetic susceptibility.