引物合成常见问题（Primer synthesis of common problems）.docVIP

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引物合成常见问题（Primer synthesis of common problems）.doc

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引物合成常见问题（Primer synthesis of common problems）

引物合成常见问题（Primer synthesis of common problems） The paper was published at 11:333-| only to see the authors introduction and common problems primer 1. How is the primer synthesized? At present, the synthesis of primers is based on the solid phase subphosphorylation. DNA synthesizer has a lot of kinds, mainly is by the ABI/PE company production, no matter use what machine synthesis, synthetic principle are the same, the main difference lies in the synthesis of high and low production rate, the difference of the reagent consumption and single cycle time. The main type of synthesizer used by shen energy gaming company is ABI3900 high flux synthesizer, and the combined growth chain is mainly using the DNA synthesizer of Beckman 1000M and PE8909, and the synthesis of primer and high OD number is ABI394. The synthesis of DNA fragments by the synthesis of phosphor triglyceride has the characteristics of high efficiency, rapid coupling and stable starting reactants. And phosphoramide three ester is fix on solid phase carrier DNA strands of DNA synthesis, synthesis of primers was synthesized from staying in the direction of the 3 end to the 5 end of synthesis, the adjacent nucleotides by 3 - 5 dihydrogen phosphate ester bond connection. ? The first step is to remove the 5 -hydroxyl group of the active group from the protective group of the active group that is prewired to the CPG of the solid phase to be protected. ? The second step, and raw material for the synthesis of DNA, and phosphoramide protect the nucleotide monomers, mixed with activator four azole nitrogen, get nucleoside phosphite activation intermediates, it is the 3 end of the activation, 5 - is still DMT hydroxyl protection, with the 5 - free hydroxyl in the solution condensation reaction. ? The third step, with cap (capping) reaction, condensation reaction may have a small number of 5 - hydroxy (less than 2%), did not participate in the reaction with acetic anhydride and 1 - methyl imidazole terminate thereafter