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Cahiers d'études et de recherches francophones / Santé

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PCR, non radioactive probes and clinical diagnosis Volume 4, issue 1, Janvier-Février 1994

Authors
Université de Corse, laboratoire de biologie moléculaire appliquée, BP 52, 20250 Corte, France, Centre hospitalier général, La Miséricorde, 20000 Ajaccio, France.
  • Page(s) : 43-52
  • Published in: 1994

Until 1985 the only way to study a gene was to clone it. Henceforth, the polymerase chain reaction (PCR) is an alternative method for synthesizing millions of copies of a specific DNA sequence. Without the development of non radioactive probes, these technologies would have been reserved for research applications. PCR with non radioactive probes is a powerful tool of molecular diagnosis in routine laboratories (identification of viruses and bacteria, diagnosis of human genetic diseases). PCR is based on Taq DNA polymerase. This enzyme is able to polymerize deoxynucleotide precursors (dNTP) in a temperature range of 75-80°C. A typical PCR reaction is a repetitive series of thermic cycles involving template DNA denaturation, oligonucleotide primer annealing, and extension of the annealed primers by DNA polymerase. This three-step process remits in the exponential accumulation of a specific fragment whose termini are defined by the 5’ end of the primers. Amplification can be estimated to be 2n, where n is the number of cycles. The first step involves denaturation of double-stranded target DNA by heating the sample to 90-95°C. In the second step, the temperature is lowered to about 5°C below the melting temperature of the primer, assuring the specificity of the primer annealing and thus the specificity of the product. The third step is carried out by raising the temperature of the sample to 70-73°C, the optimal temperature for primer extension, involving very little denaturation of the enzyme during the 25-30 cycles of a PCR reaction. The primers used are designed on the basis of the known DNA sequence and they must flank the sequence targeted. For microorganism typing a product of 300 to 900pb can be amplified, though a 2 kb product can be synthesized. The choice of the primer sequence is the function of the target and technical requirements, such as a GC content of 50-60%, which gives the optimal annealing temperature of 50-55°C. The molecular composition of the primer must be chosen to prevent the formation of intra-molecular secondary structures and primer dimers. The complementarity between the template and the 3’ OH end must be perfect, because Taq DNA polymerase activity is markedly lowered by mismatches and secondary structures. The 5’ end can thus modified by extension or base modification without altering the quality of the amplification. The yield of the reaction can be modified by the composition of the PCR medium. High concentrations of salts (MgCl2, NaCl, KCl), detergent (Laureth 12, Tween 20, SDS), chaotropic reagent (formamide), initial primer and dNTP can affect the specificity and yield. Non-radioactive labelling strategies were linked to PCR technology for medical applications. In spite of the loss of sensitivity, the advantages of cold probes are that they can be stored for over a year without signal alteration and protect laboratory staff from radiation. Two main types of labelling strategies have evolved: direct labelling in which label is attached via a covalent bond and indirect labelling, in which a hapten is attached to a base of the DNA via a spacer. In direct labelling, the label can be a chemical group (e.g sulfonation), and in the indirect procedure the most common labels are digoxigenin and biotin. Detection is carried out with an affin molecule which carries a revelation system. This molecule can be a monoclonal antibody linked to an enzyme (alkaline phosphatase or peroxidase) or a protein which binds to the hapten (e.g streptavidin-biotin). Revelation can be done directly (fluorescent chemiluminescent methods) or indirectly via an enzymatic reaction on a chromogenic substrate. Applications of cold probes in PCR have been facilitated by the availability of kits. The hybridation step of a labelled oligonucleotide probe to the amplified DNA target is still an obligation, but some new protocols circumvent this step. First, double PCR increases the amount of amplified DNA 100 times, and if the second pair of primers is chosen inside the first product, the specificity is great enough to detect bacteria or to determine the sex of a human fœtus. Direct analysis of the length or of the restriction map of the PCR product can discriminate the allelic polymorphism of a human gene or the kind of genetic mutation associated with a particular disease. While not replacing conventional methods, PCR protocols will become the method of diagnosis in routine laboratories. However, these technologies require special training and collaboration between scientists, chemists and physicians.