ALLELE-SPECIFIC PCR FOR SIMULTANEOUS AMPLIFICATION OF BOTH ALLELES OFA DELETION POLYMORPHISM IN INTRON-6 OF THE HUMAN DOPAMINE-2 RECEPTOR GENE (DRD2)

The human dopamine 2 receptor gene (DRD2) is an important candidate ge ne for drug addiction and alcoholism. So far, no mutations within the coding region of DRD2. have been found to be associated with addiction disorders. To identify sequence polymorphisms for further haplotype a nalyses and to analyze the importance of possible intron sequence vari ations of the human DRD2. gene (>260kb) in greater cohorts and in a ro utine manner we established an optimized methodological procedure for polymerase chain reaction (PCR) amplification and direct non-radioacti ve sequencing followed by a bidirectional allele-specific PCR protocol ; the latter one allows the simultaneous amplification of several alle les 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 de letion polymorphism in intron 6. The allele-specific PCR allowed the r eliable testing of 95 healthy control individuals and 270 alcoholics f or analyzing a possible genetic association of this newly characterize d polymorphic DRD2 marker with alcoholism in an ethnically and clinica lly homogenous group of patients. However, the observed allele frequen cies for the 1bp deletion polymorphism were 15.9% for the alcoholics a nd 15.3% for the controls suggesting no association of the deletion to alcoholism.