TaqMan Applications in Genetic and Molecular Toxicology

Abstract

Quantification of nucleic acids has become a common procedure in many toxicology laboratories. Among the technologies that accomplish this is the fluorogenic 5'-nuclease assay, commonly known as TaqMan. Three TaqMan applications for genetic and molecular toxicology are presented in this article: quantification of gene expression, detection of genetic polymorphisms, and quantification of chromosomal DNA deletions. Of these, quantification of gene expression is the most widely used, and established TaqMan as a benchmark technology for nucleic acid quantification. Two additional applications, polymorphism detection and quantification of DNA deletions, demonstrate the flexibility and quantitative strengths that make TaqMan so powerful, including high precision, excellent sensitivity, and broad linear dynamic range. These and similar applications improve our ability to investigate genetic and molecular dimensions of toxicological phenomena, and have promoted the widespread use of TaqMan in toxicology departments in the pharmaceutical industry. In addition to presenting these applications, the authors discuss some of the challenges of integrating TaqMan and other new technologies into the drug development process.

COMT Val158Met modulates the effect of childhood adverse experiences on the risk of alcohol dependence

“…Molecular analyses were performed in a CCKL‐accredited laboratory at the Department of Human Genetics of the Radboud University Medical Centre in Nijmegen, the Netherlands. The COMT Val158Met (rs4680) polymorphism was genotyped using Taqman‐based analysis (Watson & Li 2005). Genotyping was carried out in a volume of 10 µl containing 10 ng of genomic DNA, 5 µl of ABgene Mastermix (2x) (ABgene Ltd., Hamburg, Germany), 0.125 µl of the Taqman assay (assay ID: Taqman assay: C__25746809_50; reporter 1: VIC‐A‐allele; Applied Biosystems, Nieuwerkerk a/d Ijssel, the Netherlands) and 3.875 µl of H 2 O. Amplification was performed on a 7500 Fast Real‐Time PCR System, starting with 15 minutes at 95°C, followed by 50 cycles of 15 seconds at 95°C, 1 minute at 60°C.…”

No Evidence for the Association between a Polymorphism in the PCLO Depression Candidate Gene with Memory Bias in Remitted Depressed Patients and Healthy Individuals

“…Molecular analyses were performed in a CCKL-accredited laboratory at the Department of Human Genetics of the Radboud University Medical Centre. The PCLO rs2522833 polymorphism was genotyped using TaqMan analysis [63]. Genotyping failed for two remitted patients and nine healthy individuals, resulting in a call rate of 99.4% for remitted patients and 97.5% for healthy individuals, respectively.…”

Reduced Dopamine Receptor Sensitivity as an Intermediate Phenotype in Alcohol Dependence and the Role of the COMT Val158Met and DRD2 Taq1A Genotypes

“…The DRD2 Taq1A rs1800497 and COMT rs4680 polymorphisms were genotyped using TaqManbased analysis. 64 Genotyping was performed in a volume of 10 µL containing 10 ng of genomic DNA. For DRD2, 5 µL of Taq-Man Mastermix (2; Applied Biosystems), 0.125 µL of TaqMan assay (TaqMan assay: C_7486676_10, reporter 1, VIC-A-allele, reverse assay; Applied Biosystems), and 3.875 µL of water were added.…”