[A new approach to enhance PCR specificity].
A new approach to enhanced specificity and product yield of polymerase chain reaction is proposed. It is based on control of DNA polymerase activity during PCR by changing the magnesium ion concentration, which depends on the temperature of the reaction mixture. A slightly soluble magnesium salt, magnesium oxalate, whose solubility depends on temperature, was used as a source of magnesium ions. During PCR, magnesium oxalate was maintained at saturating concentration by the presence of an insoluble excess of this salt, and the concentration of magnesium ions depended on the salt solubility: binding of magnesium ions at lower temperatures and their release at higher temperatures was shown to affect the DNA polymerase activity and to favor the specific PCR amplification of the target DNA fragment.
Ignatov
K B
KB
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia. ignatov@ibch.ru
Miroshnikov
A I
AI
Kramarov
V M
VM
D000818:Animals; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D015236:HLA-C Antigens / Q000235:genetics; D006801:Humans; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D010070:Oxalates / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012269:Ribosomal Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D012995:Solubility; D019914:Taq Polymerase / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
D015236:HLA-C Antigens; D010070:Oxalates; D012269:Ribosomal Proteins; C029466:ribosomal protein L2; D004247:DNA; D019914:Taq Polymerase; D008274:Magnesium