DNA-based assays

Dahlback B. Resistance to activated protein C, the Arg506 to Gin mutation in the factor V gene and venous thrombosis. Functional tests and DNA based assays, pros and cons. Thromb Haemost, 1995 73,739-42. 

DNA is not tissue specific (i.e., the same DNA sequence is present in aU cells of the same organism, resulting in the impossibility of distinguishing egg from other chicken parts and milk from cattle products, including meat and entrails used as ingredients in product formulations. Additional limitations to the analysis of egg and milk by DNA-based assays are discussed in Section 9.5.5. 

Several factors affect the performance of DNA-based assays. They need to be evaluated carefully before and during the design and development of the assay. Assay performance relies on DNA integrity, DNA quality, DNA quantity and recovery, and the presence of assay inhibitors or interfering compounds. 

Basically all natural contaminants, such as food allergens or adulterating agents such as cow s milk in other species milk, can be detected by antibody-based applications as well as by alternative DNA-based assays. However, artificially synthesized components can only be determined by the use of immunoassays, since they have no DNA associated with their production. Refer to Chapter 9 for further information on DNA-based assays for food allergens. 

In general terms, DNA-based assays are easier to develop than immunoassays since the only requirements are the availability of the gene or DNA sequence to be targeted. These sequences are needed to design primers (PCR) and probes (RT-PCR) as well as for determining the G/C ratio to optimize assay conditions. This information can be obtained from databases, and software is available to facilitate the design of primers and probes. These probes have also become less expensive to produce. 

Sample preparation. Although there is not a gold standard for sample preparation and target extraction for analysis by immunoassay, most use saline buffers (e.g., phosphate-buffered saline, Tris saline buffer, high-salt buffers) at neutral pH to prevent interference and inhibition of antigen-antibody binding. Similarly, there is no standardized extraction method for DNA-based assays, and extraction efficiency is matrix dependent. However, unlike immunoassays, DNA-based assays will withstand the use of harsh conditions. 

The discovery of aptamers in 1990 has opened a new scenario in the development of RNA/DNA-based assays for different molecules (Tuerk and Gold, 1990 McGown et al., 1995), but a further advance in nucleic acid nanotechnology was made in 1994 with the first application of a DNA with a catalytic function (DNAzyme) (Breaker and Joyce, 1994). An example of DNAzyme is a single-stranded guanine-rich nucleic acid that has been reported for binding a hemin to yield a DNAzyme possessing peroxidase-like activity. It was proposed that the intercalation of hemin into... 

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