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Chapter 3. Analytical Methods to Identify Genetic Variations and Bioinformatics

Andrea Gaedigk, MS, PhD; Xia Yang, PhD

  • Define terminology for pharmacogenomic testing.
  • Outline methods used for genetic testing in pharmacogenomics.
  • Provide information on informatics in pharmacogenomics.

There are numerous methods or platforms to detect DNA sequence variations.1 These can be divided into two categories, those designed to identify specific SNPs of interest, for example, to determine a person's genotype for a given drug-metabolizing enzyme or a panel of genes, and those supporting genome-wide analyses.

Following are brief descriptions of some popular methods that are frequently used to determine a person's genotype. All depend on the polymerase chain reaction (PCR), which is not described in further detail here; abundant information can be found online or in technical guides provided by manufacturers of PCR supplies.

Restriction-Fragment Length Polymorphisms (RFLPs)

This method exploits a sequence difference between the wild-type and variant that impacts the recognition site of a restriction enzyme. The region of interest is amplified by PCR and subsequently incubated with a restriction enzyme. Resulting fragments are then separated by agarose gel electrophoresis. Figure 3–1 provides an overview. This method was one of the earliest employed for genotyping detecting SNPs. It is simple to perform and requires only basic equipment. It is, however, time consuming due to the enzymatic digestion and electrophoresis step and cannot be automated. RFLP analysis can also be performed on restricted genomic DNA to identify fragment patterns associated with a disease or a phenotype. In that case, the procedure is often referred to as Southern blot analysis.2

Figure 3–1
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Genotyping by RFLP analysis. (A) To detect the CYPD6*4 allelic variant, a 392 base pair (bp) long PCR product was amplified from genomic DNA. The wild-type allele carries two recognition sites for the BstNI restriction enzyme as indicated by scissors. The SNP characterizing the CYP2D6*4 allele destroys one of those sites as indicated by the star. Consequently, the PCR products derived from the wild-type and variant alleles will be cut into three and two fragments, respectively. (B) The resulting digestion products were separated by size using agarose gel electrophoresis, visualized with ethidium bromide, a DNA-intercalating dye, and scanned for documentation. Subjects homozygous for the wild-type allele exhibit 192, 161, and 39 bp long fragments, subjects homozygous for CYP2D6*4 have 363 and 39 bp long fragments, and heterozygous subjects exhibit a composite pattern. Genotypes of the subjects are as indicated. M denotes the 100 bp marker reference ladder.

Real-Time PCR

In real-time (RT) PCR, the formation of amplification product is detected at each cycle. This can be achieved by measuring fluorescent dyes that intercalate into DNA such as SYBR green,3 fluorogenic-labeled probes such as TaqMan chemistry3 (also known as “fluorogenic 5” nuclease chemistry), or molecular beacons,4 to name a few. TaqMan® SNP Genotyping Assays are frequently utilized for ease of use and ...

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