Phosphoramidite monomer composition and preparation method and application thereof

Abstract

The invention provides a phosphoramidite monomer composition and a preparation method and application thereof. The phosphoramidite monomer composition comprises a dA solution, a dC solution, a dG solution and a dT solution, wherein the concentration of the dA solution is 0.01-0.05g / mL; the concentration of the dC solution is 0.01-0.03g / mL; the concentration of the dG solution is 0.01-0.03g / mL; the concentration of the dT solution is 0.01-0.03g / mL; and the volume ratio of the dA solution to the dC solution to the dG solution to the dT solution is (5-6) to (4-5.1) to (4-5.1) to (4-5). The phosphoramidite monomer composition provided by the invention is reasonable in proportion; the distribution of degenerate sites in prepared oligonucleotide is reasonable; and the degeneracy of each degenerate site is within the range of 20-30%.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid synthesis, and relates to a phosphoramidite monomer composition and its preparation method and application, in particular to a phosphoramidite monomer composition and its preparation method and its use in the synthesis of degenerate sites Applications in oligonucleotides. Background technique [0002] Short-chain DNA, also known as oligonucleotide (Oligo), is usually synthesized by efficient and automated solid-phase synthesis technology. Solid-phase synthesis uses controlled pore glass spheres (CPG) as the solid-phase carrier, and four phosphoramidites A, T, C, and G as monomers, and undergoes deprotection, condensation, oxidation, and capping according to the 3'-5' direction The cycle of four reactions gradually ligated the target sequence on the solid phase support (Andrew Ellington et al., Introduction to the Synthesis and Purification of Oligonucleotides, Current Protocols in Nucleic Ac...

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Problems solved by technology

However, limited to the technical level at the time of literature reports, the reactivity of the four monomers is mainly detected based on HPLC analysis, proton nuclear magnetic resonance spectrum analysis or some biological methods. These methods cannot directly characterize the degeneracy of the synthetic sequence, that is, they cannot directly "Reading" oligos with degenerate sites, and testing only for oligos with a few deoxynucleotide sites, resulted in four monomers with different activities than those with more degenerate sites, longer chains Oligo, so the results are not convincing

According to the ratio of phosphoramidite reported in the prior art, the four monomers are mixed, and the degeneracy of the synthesized Oligo degenerate N deviates greatly from 25%.

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