[Polymerase chain reaction, cold probes and clinical diagnosis].
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 degrees 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 results 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 degrees C. In the second step, the temperature is lowered to about 5 degrees 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 degrees 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 a function of the target and technical requirements, such as a GC content of 50-60%, which gives the optimal annealing temperature of 50-55 degrees 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Haras
D
D
Université de Corse, laboratoire de biologie moléculaire appliquée, Corte, France.
Amoros
J P
JP
D001483:Base Sequence; D015342:DNA Probes; D004259:DNA-Directed DNA Polymerase; D005820:Genetic Testing; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D013696:Temperature
D015342:DNA Probes; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase
Réaction de polymérisation en chaîne, sondes froides et diagnostic clinique.