Apolipoprotein E genotyping method by real time PCR, a fast and cost-effective alternative to the TaqMan and FRET assays.
The apolipoprotein E gene (APOE) polymorphism genotyping has an allegedly important predictive value for coronary heart disorders and Alzheimer's disease. We developed a simple, fast, cost-effective and suited for high-throughput protocol for determining APOE genotypes by Real Time PCR monitored by SYBR Green. The method is based on differential amplification by allele-specific primers. These primers have variations in their 3'-end nucleotides such that are specific for one of the two variants in each polymorphic position. By this protocol, we obtained a 100% concordance with the APOE genotypes determined by sequencing analysis. The main advantages of this method are its relative simplicity and the reduced cost compared to other methodologies, such as the TaqMan and FRET assays.
Calero
Olga
O
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain.
Hortigüela
Rafael
R
Bullido
María J
MJ
Calero
Miguel
M
D001057:Apolipoproteins E / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D019914:Taq Polymerase
D001057:Apolipoproteins E; D019914:Taq Polymerase