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CHRISTINA L. AQUILANTE, PHARM.D., is Assistant Professor, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado at Denver and Health Sciences Center, Denver. ISSAM ZINEH, PHARM.D., is Assistant Professor, Department of Pharmacy Practice, College of Pharmacy, University of Florida (UF), Gainesville. AMBER L. BEITELSHEES, PHARM.D., is Research Assistant Professor, School of Medicine, Washington University, St. Louis, MO. TAIMOUR Y. LANGAEE, PH.D., M.S.P.H., is Research Assistant Professor, Department of Pharmacy Practice, College of Pharmacy, and Director, Genotyping Core Laboratory, Center for Pharmacogenomics, UF.

Address correspondence to Dr. Aquilante at the Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado at Denver and Health Sciences Center, 4200 East Ninth Avenue, Box C238, Denver, CO 80262 (christina.aquilante{at}uchsc.edu).

Purpose. Common laboratory methods used in pharmacogenomics studies are described. Summary. The reliable and accurate determination of a person’s genetic makeup at a particular locus in the DNA molecule, or genotype, is fundamental to pharmacogenomics. Whole blood cells and buccal cells are commonly collected to obtain a DNA sample. Once DNA is collected, the genomic DNA must be isolated from other cellular material. Next, a specific region of interest must be identified and amplified, performed via polymerase chain reaction (PCR). Gel electrophoresis is often performed after PCR to verify that PCR was successful and that the amplified target sequence is the correct size. Numerous methods are available to determine a person’s genotype and differ based on allele discrimination and detection. PCR coupled with restriction fragment length polymorphism (RFLP) analysis, a conventional genotyping method, does not rely on automated technology and is practical for laboratories that genotype a limited number of samples. Pyrosequencing is an automated genotyping method in which the principal allele discrimination method is a primer extension reaction coupled with a luciferase-based enzyme reaction. TaqMan relies on the use of fluorescencelabeled probes, in addition to PCR primers, in the reaction mixture, enabling PCR amplification and allele discrimination in the same step. Mass spectrometry differentiates DNA molecules using a defined mass. Denaturing high-performance liquid chromatography (DHPLC) uses a reverse-phase ion-pair column to discriminate between variant and nonvariant alleles. Conclusion. An understanding of the common genotyping methods used in pharmacogenomics studies, including PCR-RFLP analysis, pyrosequencing, TaqMan, mass spectrometry, and DHPLC, will aid pharmacy practitioners and students when interpreting the methods sections of such studies. Index terms: Analysis; Methodology; Pharmacogenetics; Tests, laboratory

 

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