[A rapid and high throughput method for apoE genotyping].
OBJECTIVE
To develop a new method that can determine the apolipoprotein E(apoE) genotypes rapidly in high throughput.


METHODS
Genome DNA samples were extracted from the anticoagulated peripheral blood samples of 79 patients with Alzheimer's disease(AD) and 63 healthy individuals, and the 492 bp apoE gene fragments including 112 and 158 codons were amplified by polymerase chain reaction (PCR). With one PCR product, three recombined alleles (epsilon 2, epsilon 3 and epsilon 4) of apoE gene as controls were obtained by cloning and site-directed mutagenesis. The excess primers and dNTPs in all PCR products were removed by treatment with clean up reagents, then template-directed dye-terminator incorporation reaction (TDI) was performed and R110 or TAMRA labeled Acyclo-terminators were added into the mutation sites specifically. Fluorescence polarization value (FP) was measured using victor 2 multilabel counter and the polymorphisms in 112 and 158 condons of apoE gene were investigated.


RESULTS
The apoE genotypes in recombined plasmid controls and all serum samples were analyzed using the authors' TDI-FP method, and the reliability and specificity were confirmed by DNA sequencing. The frequency of epsilon 4 allele in patients was significantly higher than that in controls, suggesting that apoE epsilon 4 allele gene is a risk factor for late-onset AD.


CONCLUSIONS
TDI-FP is an easy, reliable and high throughput technology in analyzing polymorphism of apoE gene; it can be used in the prediction of susceptibility to AD in elderly individuals. Furthermore, it is an ideal method for large-scale screening and for studying the relationship between the allelic and genotypic frequencies of apoE and other diseases.
Zhang
Wen-hong
WH
Institute of Gene Diagnostic Technique of Chinese PLA, Fourth Military Medical University, Xi'an, Shaanxi, 710032 PR China.
Bai
Yu-jie
YJ
Wang
Yan
Y
Li
Ding
D
Yan
Xiao-jun
XJ
D000368:Aged; D000483:Alleles; D000544:Alzheimer Disease / Q000235:genetics; D001057:Apolipoproteins E / Q000235:genetics*; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results
D001057:Apolipoproteins E