Genotyping and haplotyping of the dopamine D4 receptor gene by capillary electrophoresis.

In this paper we report on simultaneous genotyping of adjacent polymorphisms (referred to as haplotyping) by combining double-tube allele-specific polymerase chain reaction, restriction fragment length polymorphism and capillary gel electrophoresis analysis of the resulting fragments. Direct molecular haplotyping is of particular importance in the case of double heterozygote samples, since in these instances the haplotype structure cannot be constructed based on genotype data. Our approach provided a powerful tool for coincidental genotype analysis of the 48 base pair (bp) variable number of tandem repeats of the third exon and haplotype investigation of the -616CG and -521CT single nucleotide polymorphisms of the dopamine D4 receptor (DRD4) gene. The linear polyacrylamide sieving matrix was optimized for the size range of the double-stranded DNA fragments of interest varying from 35 to 763 bp. We demonstrated that capillary gel electrophoresis in combination with laser induced fluorescence detection offers a sensitive and accurate tool for automated haplotyping in clinical settings.