Fig

FIGURE 24.23 Zipcoded SNP Analysis by Single Base Extension

A segment of DNA that includes an SNP site is generated by PCR (only a single strand of the DNA is shown here, for simplicity). Single base extension is performed with a primer that binds one base in front of the SNP. Person I has an A at the SNP site and therefore T is incorporated; in person II, a G results in incorporation of C. The incorporated bases are labeled with different fluorescent dyes. The elongated primer is then trapped by binding of its Zipcode sequence to the complementary cZipcode, which is attached to a bead or other solid support.

SNP analysis looks for single base changes in critical regions of the genome.

SNP analysis is used increasingly to screen for possible hereditary defects and also to test for individual variation in genes that affect the response to pharmaceuticals. A variety of methods are in use, but most start by using PCR to generate the region of DNA that contains the polymorphism. The sequence must then be determined—but only for a single base at one precise location. Thus it is not necessary to sequence the whole PCR fragment. Instead a single base extension reaction is performed, using a primer that binds just in front of the polymorphism site plus specifically labeled dideoxynucleotides. Thus each of A, T, G and C could be labeled with fluorescent dyes of different colors. Using dideoxynucleotides ensures that the primer is only elongated by a single base—the one that is complementary to the base at the polymorphism site. The elongated primer will now be fluorescently labeled, and its color will reveal which base was present in the SNP (Fig. 24.23).

Pharmacogenomics—Genetically Individualized Drug Treatment

The genetic differences between individuals may cause significant differences in their reactions to certain drugs or clinical procedures. Cytochrome P450 plays a major role in the oxidative degradation of many foreign molecules, including a wide range of pharmaceuticals. Cytochrome P450 is actually a family of several closely related enzymes, whose substrate range varies so providing protection against a wide range of different foreign molecules. For example, the CYP2D6 isoenzyme oxidizes drugs of the tricyclic antidepressant class. Any given cytochrome P450 may have multiple allelic variants, some of which may show altered activity. Thus the CYP2D6 gene has several alleles with lowered activity and also one (a duplication) with increased activity (Table 24.02). Such alleles may be present at different frequencies in different human populations. Patients who possess low activity alleles metabolize the corresponding drugs much more slowly and are consequently not only more sensitive to the desired effects of the drug but also more likely to show toxic side effects. In such cases, individual SNP analysis of patients can reveal which allele is present before administering the drug. The drug dosage can then be adjusted to the individual patients genetic constitution. The new and rapidly expanding field that relates individual genotypes to pharmaceutical treatment is known as pharmacogenomics.

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