High-throughput chemiluminometric genotyping of single nucleotide polymorphisms of histamine, serotonin, and adrenergic receptor genes.
Several pharmacogenetic studies are focused on the investigation of the relation between the efficacy of various antipsychotic agents (e.g., clozapine) and the genetic profile of the patient with an emphasis on genes that code for neurotransmitter receptors such as histamine, serotonin, and adrenergic receptors. We report a high-throughput method for genotyping of single nucleotide polymorphisms (SNPs) within the genes of histamine H2 receptor (HRH2), serotonin receptor (HTR2A1 and HTR2A2), and beta(3) adrenergic receptor (ADRB3). The method combines the high specificity of allele discrimination by oligonucleotide ligation reaction (OLR) and the superior sensitivity and simplicity of chemiluminometric detection in a microtiter well assay configuration. The genomic region that spans the locus of interest is first amplified by polymerase chain reaction (PCR). Subsequently, an oligonucleotide ligation reaction is performed using a biotinylated common probe and two allele-specific probes that are labeled at the 3' end with digoxigenin and fluorescein. The ligation products are immobilized in polystyrene wells via biotin-streptavidin interaction, and the hybrids are denatured. Detection is accomplished by the addition of alkaline phosphatase-conjugated anti-digoxigenin or anti-fluorescein antibodies in combination with a chemiluminogenic substrate. The ratio of the luminescence signals obtained from digoxigenin and fluorescein indicates the genotype of the sample. The method was applied successfully to the genotyping of 23 blood samples for all four SNPs. The results were in concordance with both PCR-restriction fragment length polymorphism analysis and sequencing.
Toubanaki
Dimitra K
DK
Department of Chemistry, University of Patras, 26500 Patras, Greece.
Christopoulos
Theodore K
TK
Ioannou
Penelope C
PC
Flordellis
Christodoulos S
CS
D005838:Genotype; D006801:Humans; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D022702:Receptors, Adrenergic, beta-3 / Q000235:genetics*; D011970:Receptors, Histamine H2 / Q000235:genetics*; D011985:Receptors, Serotonin / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
D022702:Receptors, Adrenergic, beta-3; D011970:Receptors, Histamine H2; D011985:Receptors, Serotonin