MadSci Network: Molecular Biology

Re: what is the process of DNA polymerase primer synthesis in simple terms?

Date: Thu Mar 27 16:58:42 2003
Posted By: Daniel Lafontaine, Scientist
Area of science: Molecular Biology
ID: 1045035326.Mb

Dear Lynsey,

You are basically asking about chemical synthesis of DNA. This is usually achieved using an
apparatus called "DNA synthesizer". You can see one of them here. This idea is that wich the
proper chemicals, one is able to make any possible DNA sequences from 1 to just about 150 nucleotides. Scientists tend to buy them already made from external companies (Invitrogen and Sigma-Aldrich are some of them). However, as you were wondering, there is some good chemistry involved in the making of DNA oligos and this is were we are going now. First of all, the synthesis of any oligos is made from the 3' end of the sequence to the 5'end (we use the notation 3'->5'). So, for each phosphoramidites (which are the nucleosides equipped with protecting chemical groups) added, there is 4 steps: 1)Detritylation 2)Coupling 3)Capping 4)Oxidation The first phosphoramidite of the 3'end is always attached to a column and therefore, the resulting DNA oligos will also be attached to this column. Also, the first phosphoramidite is protected with some chemical groups (DMT) in order to be more stable and inert. In order to react with the second phosphoramidite, one must remove these chemical group and this is the Detritylation step. Next, the second phosphoramidite is introduced and can now be attached to the first one. This is the Coupling step. In fact, we need to activate the second phosphoramidite but I am not covering this here since we are just talking about the main steps. So now, we have to think about something: there will be some couplings that will not work. The net result will be that there will be a mixture of one and two residue molecules. The one residue molecules are the ones where the coupling didn't work. Since these molecules could in principle be coupled to the third phosphoramidite added (and thus changing the resulting DNA sequence), we need to prevent them to react. This is the Capping step. This is like a protection system that will ensure that if one coupling did not work, the resulting sequence will not be the same length as the good one. These days, it is easy to purify DNA oligos that have different lengths. The last step is the Oxidation. During the coupling reaction, the phosphate is the reactive group and this step is to convert the phosphate to what is found in the DNA backbone (+5 valence form). Once the oxidation is completed, the process can start over again with the next addition. At the end of the synthesis, some blocking groups (on the A,C,T and G bases) need to be removed, the DNA oligos can be cleaved from the support and be purified by various methods like the electrophoresis or HPLC. Hope this helps and you can have a little scheme of this discussion here. Dan

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