The development of polymerase chain reaction (PCR) technique. The research by collaborators revolutionized molecular biology and molecular medicine. Polymerase chain reaction is a method used for amplification by enzymes of a specific region of DNA that is located between two regions of a known DNA sequence. While in the past only very small amounts of a particular gene could be obtained. What is pcr used for. PCR now allows you to amplify even one copy of a gene to a million copies in a few hours.
What reagents are needed for a typical polymerase chain reaction (pcr)? PCR methods have become indispensable for many general procedures, such as cloning specific DNA fragments, detecting and identifying genes for diagnostic and forensic purposes, and researching expression methods. gene expression More recently, PCR has allowed research into new areas, such as food authenticity control, the presence of genetically modified DNA and microbiological contamination. To understand the principles of PCR and its application, you must first study the nature of the DNA molecule. This is why the structure and replication of DNA is described in the next section.
The presence of divalent cations in PCR is significant. The concentration of MgCl 2 in the final reaction mixture is usually from 0.5 to 5.0 mm, and the optimal concentration is determined empirically. Mg2 + ions: form a soluble complex with dNTPs, which is necessary for the inclusion of dNTPs, stimulate polymerase activity, increase the Tf primer / template interactions (and thus stabilize the interaction between the two chains). As a rule, a low concentration of Mg2 + leads to a low yield (or to zero production), while a high concentration of Mg2 + leads to the accumulation of non-specific products (“mesam”). It is important to avoid the presence of high concentrations of chelators, such as EDTA, or negatively charged ionic groups, such as phosphate, in the DNA matrix solution. Various buffers and additives for PCR, such as DMSO, PEG 6000, formamide, glycerin, spermidine and nonionic detergents, which are used to increase the specificity or efficiency of the reaction, are discussed in the current literature. Some DNA polymerases actually achieve their optimal level of activity only in the presence of these additives.
At this stage, the primers extend to the target sequence using thermostable DNA polymerase (often Taq DNA polymerase) in the presence of dNTP, which leads to duplication of the original target material. The ideal working temperature for Taq DNA polymerase is 72 ° C. When the primers are expanded on several bases, they have a stronger ionic attraction for the matrix, which reduces the likelihood of the reverse process. Primers that do not exactly match will come off again (due to a higher temperature) and will not lead to fragment expansion. Bases (optional to the template) are linked to a 3 ‘primer (polymerase adds 5’ to 3 ‘dNTPs, reading 3’ to 5 ‘template). The duration of the primer extension steps can be increased if the amplified DNA region is long; however, for most PCR reactions, an elongation time of 1 minute is sufficient to achieve full elongation.
This method establishes general principles for screening or identification tests for plant pests using PCR and related methods. Formal methods specific to the matrix / pest pair or, if this is not the case, suppliers of reagents, determine specific additional requirements that must be implemented for the practical implementation of the analysis, in particular: – the nature and preparation of the sample for analysis; – the nature and methods of analysis; methods for the isolation and purification of DNA or RNA (fragmentation, grinding, maceration, etc.); – mix composition and amplification program; – methods of preserving plant material and extract. Special instructions can also establish conditions or elements that can affect the quality of the results, such as PCR reagents: – general atmosphere in the room (temperature, cleanliness), “know-how” (pipetting, plate processing, gel quality). agarose for conventional PCR, image capture device on gels, etc.) – threshold values selected for interpretation and background noise (real-time PCR).
In all cases where technical requirements differ between sources, laboratories should use specific official methods, general official methods or, in the absence of other elements, suppliers in order of priority. In case of doubt, the laboratory can always seek the opinion of the national reference laboratory corresponding to the relevant line of analysis.