Genomic DNA for genotyping was extracted from total blood samples collected in ethylenediaminetetraacetic acid (EDTA) (Wizard Genomic DNA Purification Kit: Promega®, USA) and kept frozen at seventy degrees Celsius negative (-70ºC) until analysis. For APOE genotyping (alleles Ɛ2, Ɛ3 and Ɛ4), DNA samples were amplified by polymerase chain reaction (PCR), followed by digestion with Haemophilus haemolyticus restriction endonuclease I (HhaI) and restriction fragment length polymorphism (RFLP) analysis, as previously described by Hixson and Vernier.
The CYP2D6\*3, CYP2D6\*4 and CYP2D6\*6 alleles were investigated by tetra-primer PCR and the CYP2D6\*5 allele, which presents the entire CYP2D6 gene deletion, was identified by long PCR reaction, according to the method described by Hersberger et al. The CYP2D6\*10 allele was amplified by PCR following Baclig et al. and the RFLP analysis was performed with Type II restriction enzyme (HphI). For all analysis a positive and negative controls were included.
Subjects carrying Epsilon 3, Epsilon 3 (Ɛ3Ɛ3), CYP2D6\*3/4/5/6/10-nor specific mutations are classified as wild-type and functional allele carriers. The subjects carrying two defective alleles were classified as poor metabolizers (PM) and those heterozygous were classified as extensive metabolizers (EM).
