Chapter: Pharmaceutical Biotechnology: Fundamentals and Applications - Oligonucleotides

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Diagnostic Applications

Apart from the direct application of ONs in medicine as therapeutics, they also fulfill an increasingly important role as diagnostic agents.

DIAGNOSTIC APPLICATIONS

Apart from the direct application of ONs in medicine as therapeutics, they also fulfill an increasingly important role as diagnostic agents. The highly specific binding of complementary oligonucleotide sequences can be used to detect gene expressionprofiles and mutations, whereas aptamers can be used to detect presence of specific compounds. PCR amplification of specific nucleic sequences can pro-vide information on the presence and abundance of this particular sequence. For example, the Amplicor HIV-1 Monitor v1.5 assays manufactured by Roche Diagnostics are currently approved for in vitro diag-nostic use to determine viral load in blood samples.

 

Other applications, like microarrays, do not focus on single genes but provide an overview of the “transcriptome” (Fig. 8). A DNA microarray consists of ONs of approximately 25 bases that are spotted on a chip in an orderly arrangement, representing the genes of interest of an organism. Each ON is spotted at a specific location on the array so that the location of each ON with corresponding gene is known. Robotic spotters can currently place tens of thousands of ONs accurately on one slide of a few square centimeters. Each spot contains identical single-stranded ONs that are strongly attached to the slide surface, allowing cellular DNA or RNA to be labeled and hybridized to the complementary sequence on the array. By quantify-ing the binding of the labeled DNA or RNA to the specific spots, the abundance of each species can be determined and related to the corresponding gene.

 


In 2004, the FDA approved the first microarray AmpliChip CYP450 for clinical use. The AmpliChip CYP450 provides complete coverage of the gene variations, including duplications and deletion, of the cytochrome P450 enzymes 2D6 and 2C19. These genes are involved in the metabolism of approximately 25% of all prescription drugs. It could be regarded as an important step towards personalized medicine.

 

The inherent specificity and selectivity of aptamers makes these ONs very useful to detect disease-associated molecules, in a similar manner as antibody-based immunoassays. However, they are not in clinical use yet.

Padlock probes can also be used for diagnostic applications (Nilsson et al., 1994). They consist of long ONs, whose ends are complementary to adjacent target sequences. Upon hybridization, the ends of the ONs are brought together, allowing ligation of the ON ends into a closed and intertwined circle that cannot be replaced by the complementary DNA strand. This closed circle can then be amplified by rolling circle amplification, a powerful and robust DNA amplifica-tion method based on the mesophilic Phi29 DNA polymerase, allowing amplification of the padlock signal to detectable levels (Fig. 9). Padlock probes are more specific than conventional antisense ONs, as misannealing of either one of the ends does not result in proper ligation of the ends, and thus prevents circularization of the padlock probe. It has been used for multiplex detection of pathogens in biological samples (Szemes et al., 2005), single nucleotidepolymorphisms (Bakht and Qi, 2005) and miRNA (Jonstrup et al., 2006), but also for in situ genotyping of individual DNA molecules (Larsson et al., 2004).






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