Allele-specific PCR for simultaneous amplification of both alleles of a deletion polymorphism in intron 6 of the human dopamine 2 receptor gene (DRD2)

Abstract

Database Accession No. 229557–9, 229564, 229566

The human dopamine 2 receptor gene (DRD2) is an important candidate gene for drug addiction and alcoholism. So far, no mutations within the coding region of DRDZ have been found to be associated with addiction disorders. To identify sequence polymorphisms for further haplotype analyses and to analyze the importance of possible intron sequence variations of the human DRD2 gene (>260kb) in greater cohorts and in a routine manner we established an optimized methodological procedure for polymerase chain reaction (PCR) amplification and direct non-radioactive sequencing followed by a bidirectional allele-specific PCR protocol; the latter one allows the simultaneous amplification of several alleles in one reaction tube. Overall, the sequences of the DRD2 introns 3–7 are highly conserved. Nevertheless, in each of the analyzed intron sequences we found substitution variants as well as a one base-pair deletion polymorphism in intron 6. The allele-specific PCR allowed the reliable testing of 95 healthy control individuals and 270 alcoholics for analyzing a possible genetic association of this newly characterized polymorphic DRDZ marker with alcoholism in an ethnically and clinically homogenous group of patients. However, the observed allele frequencies for the lbp deletion polymorphism were 15.9% for the alcoholics and 15.3% for the controls suggesting no association of the deletion to alcoholism.