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title What's wrong with sequencing finding mutations in primers?

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Provider: Shanghai huding Biotechnology Co., Ltd Release time: July 23, 2020 reading times: 35 times >>Enter the company's stand
Sequencing found that there are mutations in the primer region, especially for primers with less than 40 bases, which is not likely to happen, but it will also happen. There are many explanations for this mutation, and there is no way to solve this problem thoroughly. The principle of solid-phase synthesis of primer synthesis is the same, and the machines used are basically the same. The main raw materials for the synthesis are provided by several multinational companies. The problems encountered by all synthesis service providers are basically similar, and no one can escape from it.

Primer synthesis is a multi-step chemical reaction, the highest synthesis efficiency is 99%, and the by-products can not be avoided. The insertion mutation in primer sequence is often base duplication. It is generally believed that in the process of coupling, DMT is lost in some of the even linked monomers, resulting in the monomers joining again, so the mutation of inserting the same base occurs. As for deletion mutation, it is generally believed that the deletion mutation is caused by incomplete capping reaction. Capping reaction mainly blocks a small number of 5 '- hydroxy groups and does not participate in the reaction monomer. The closed primers will not continue to participate in the synthesis in the next round of coupling. For base substitution mutation, it is generally believed that the base can not be 100% deprotected, that is, there may be residual protective groups on the primer, and these regions of the primer can not be well paired with the complementary chain. When the amplified product is subcloned into E.coli, the non paired base may be added by the repair system in the bacteria. Substitution mutations usually occur when G is converted to other bases. Under certain conditions, base G can be transformed into enol isomer (deproteinization), 2,6 diaminopurine. In the process of DNA replication and amplification, 2,6 diaminopurine is regarded as base a by DNA polymerase, and G-A substitution will be found in sequencing. The frequency of deproteinization was higher in purine rich primers. If the deproteinized primer is degraded during the deprotection stage of primer post-processing, the deletion of base g or a will be found by sequencing.

In the process of primer synthesis, the factors causing base insertion, deletion and substitution mutation exist objectively. There are many suggestions and measures to reduce the occurrence frequency, but these measures are still in the laboratory stage and have not been applied to large-scale production.


key word: Mutation of primers was found by sequencing ??

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