From Single Cell Gene-based Diagnostics to Diagnostic Genomics: Current Applications and Future Perspectives

Clinical Laboratory Science, Fall 2005 by Zhao, Richard

GENE-BASED AMPLIFICATION TECHNOLOGIES

The gene-based amplification technologies can be further divided into PCR-based or non-PCR based methods. PCR-based method is an artificial DNA amplification method that is performed at various temperatures using a thermocycler. In contrast, most of the non-PCR methods take advantage of the natural nucleic acid amplification processes. For example, ligase chain reaction (LCR) mimics the enzymatic ligation process; nucleic acid sequence based amplification (NASBA) mimics viral RNA reverse transcription and transcription; strand displacement assay (SDA) resembles the DNA excision repair process; and Qβ-replicase RNA amplification resembles bacteriophage replication. Another common feature of non-PCR-based assays is that these assays can be carried out at constant temperature without thermocycling.

In theory, a single molecule can be detected and amplified to 109 to 1012 molecules within a few hours of the amplification reaction. In clinical practice, however, a much lower efficiency is obtained due to impurities and other intrinsic factors within the clinical specimens that may affect the efficiency of the amplification. Nevertheless, 10 to 100 copies of the target nucleic acid sequences, for example, can normally be detected with high probability.

DETECTION OF AMPLIFIED GENE PRODUCTS

Typically the amplified gene products can be detected by using various labeled reporter molecules such as enzymes, antigenic substrates, radioisotopes, chemiluminescent moieties, or fluorescent labels (Table 1). The classical means of gene detection is the use of DNA probes labeled with radioactive ^sup 32^P. Binding of the probe to the amplified product (hybridization) enables visualization of the gene product either by autoradiography or liquid scintillation counting. In most of the current commercially available diagnostic kits, however, radioactive isotopes are replaced by enzymes, affinity labels, chemiluminescent molecules, or fluorescent reporters for the detection of the amplified products. For example, direct labeling of oligonucleotides with the enzymes alkaline phosphatase or horseradish peroxidase, is responsible for the signal amplification in the bDNA signal amplification assay. Addition of enzyme specific substrates such as dioxetane to the amplified product gives light emission that can be detected by luminometry. Affinity labels such as biotin and digoxigenin are incorporated into the primers in PCR-based assays by enzymatic (nick translation or random-priming techniques) or nonenzymatic, e.g., photobiotin methods. Streptavidin linked to an enzyme is used to detect the moiety attached to the primers, and the detection is completed through the use of colorimetric or chemiluminescent substrates for the enzyme. Chemiluminescent markers are chemical groups that release light when exposed to certain substrates after the hybridization reaction is complete. The light emitted can be detected with X-ray film or a luminometer. Such assays have sensitivity ranges equal to or greater than those of ^sup 32^P.