Rapid and direct detection of apolipoprotein E genotypes using whole blood from humans.
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.
Kim
Suhng Wook
SW
College of Health Sciences, Korea University, Seoul.
Heo
Ji Hye
JH
Kim
Chun Huem
CH
Yoo
Dong Chul
DC
Won
Dong Hwan
DH
Lee
Seung Gwan
SG
Cho
Kyoung Jin
KJ
Song
Jung Han
JH
Park
Su Jeong
SJ
Yang
Young Geun
YG
Choi
Dal Woong
DW
D000368:Aged; D000369:Aged, 80 and over; D001057:Apolipoproteins E / Q000097:blood / Q000235:genetics*; D004247:DNA / Q000097:blood / Q000235:genetics; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
D001057:Apolipoproteins E; D020033:Protein Isoforms; D004247:DNA