PMC:7143804 / 10922-13453
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7143804","sourcedb":"PMC","sourceid":"7143804","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7143804","text":"1.3. Multiple Displacement Amplification\nThe proof-of-principle amplification of choice is a MDA reaction, which is a non-specific isothermal method of amplification performed around 30 °C [50]. MDA is a method of whole genome amplification (WGA), as it amplifies all present DNA [52]. It is commonly used when the initial amount of DNA sample is very low. After the WGA is performed, a sequence specific amplification can be done since the quality the amplified DNA by MDA is very high [53]. The amplification reaction is illustrated below in Figure 1 (the contour of the amplified sequence is highlighted in black for clarity). Starting with a double stranded DNA (dsDNA) molecule, a denaturation step at 95 °C is required, giving the random hexamer-primers and the ϕ29 DNA polymerase access to the bases of single stranded DNA (ssDNA) strands. The hexamers anneal themself to aleatory parts of the ssDNA sequence. These hexamers work as initiation sites for the ϕ29 DNA polymerases. After denaturation at 95 °C, the mixture is cooled down to ice temperature and the rest of the reagents are added. The mixture is heated up to ~30 °C so the polymerase starts to complete the complementary ssDNA sequence, creating again a dsDNA strand, eventually it encounters a hexamer from another annealing site. Once this happens the polymerase will lift up that hexamer and starts to separate the amplified sequence formed from that annealing site. As the polymerase displaces the formed strand ahead of it, it continues to complete the sequence. The displaced strand becomes a new ssDNA strand and therefore, it gives new sites for more primers to attach and initiation sites for the polymerase, continuing the amplification, and thus creating a web of DNA strands. Finally, the inactivation of the polymerase is done by heating up the system to 65 °C.\nEven though MDA is considered an isothermal process, prior to the reaction and to the addition of most reactants, the dsDNA and a buffer are heated up to 95 °C to denature the dsDNA to ssDNA and to give hexamers the initial access to the ssDNA. After the amplification reaction, the polymerase has to be inactivated at 65 °C. However, this does not require fast temperature changes, as would be the case with, for example, the temperature cycling in PCR amplifications. This, together with the robustness of the amplification (it is a self-limiting reaction that amplifies all present DNA [50]) makes MDA perfectly suitable as proof-of-principle amplification reaction for such 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