DNA synthesis technology introduction
We're Professional Supplier Extract Powder manufacturers and suppliers in China specialized in providing high-quality products at low price. We warmly welcome you to buy or wholesale bulk Supplier Extract Powder for sale here from our factory. For a free sample, contact us now.
Supplier Extract Powder,Supplier Extract ,Supplier Powder Manufacturer in China Shaanxi Kang New Pharmaceutical co., Ltd. , https://www.kangnewpharmas.com
DNA synthesis: A method in which deoxynucleotides are artificially linked one by one to synthesize a DNA strand in the order of a predetermined nucleotide. At present, solid phase synthesis is used, that is, the nucleotide is extended from the 3' end on the polymer support, and can be automated. )
At present, oligo DNA synthesis generally uses solid phase phosphoramidite triester method to synthesize DNA fragments. This method has the advantages of high efficiency and rapid coupling, and has been widely used in DNA chemical synthesis. DNA chemical synthesis differs from the enzymatic DNA synthesis process in that it extends from the 5'®3' direction, but starts at the 3' end.
The specific reaction steps are as follows:
1. Deblocking: The protective group DMT (dimethoxytrityl) of the nucleotide attached to CPG (Controlled Pore Glass) was removed by using Trichloroacetic Acid (TCA). Free 5'-hydroxyl end for the next condensation reaction.
2. Activation: The phosphoramidite-protected nucleomonomer is mixed with the tetrazole activator and introduced into the synthesis column to form a phosphoramidite tetrazole active intermediate (the 3'-end has been activated, but The 5'-end is still protected by DMT) and this intermediate will undergo a condensation reaction with the deprotected nucleotide on the GPG.
3. Coupling: When a phosphoramidite tetrazole active intermediate encounters a deprotected nucleotide on CPG, it will react with its 5'-hydroxyl group, condense and remove the tetrazole, and synthesize it. The oligonucleotide strand is extended forward by one base.
Capping: After the condensation reaction, in order to prevent the 5'-hydroxyl group which is not involved in the reaction on the CPG from being extended in the subsequent cyclic reaction, the terminal hydroxyl group is often blocked by acetylation, and the general acetylating reagent is It is formed by mixing acetic anhydride and N-methylimidazole.
5. Oxidation: During the condensation reaction, the nucleotide monomer is linked to the oligonucleotide attached to the CPG through a phosphorous ester bond, and the phosphorous ester bond is unstable, and is easily hydrolyzed by an acid or a base. The phosphorous acid is converted to a phosphate triester by a solution of iodine in tetrahydrofuran to obtain a stable oligonucleotide.
After the above five steps, a deoxynucleotide is attached to the nucleotide of the CPG, and the protective group DMT on the newly added deoxynucleotide 5'-hydroxyl group is also removed by trichloroacetic acid. The above DNA fragmentation can be obtained by repeating the above activation, ligation, blocking, and oxidation processes.
Finally, it is cleaved and deprotected (generally protected with benzoyl group for A and C bases; protected with isobutyryl group for G base; T base is not protected; nitrile is protected with nitrile ethyl), purified (commonly used The HAP, PAGE, HPLC, C18, OPC, etc. methods, quantitative and other synthetic post-treatment can obtain oligonucleotide fragments that meet the experimental requirements.
The Oligo synthesized by the above method polypeptide has a very low purity of the target Oligo after deprotection, and contains a large amount of impurities. The main impurities are: benzoic acid ammonia and isobutyric acid ammonia formed by the protective group which is removed, a nitrile ethyl group which is removed from the nitrile phosphorus group, and a short chain which is produced during synthesis. As a result, the full-length Oligo DNA content in the crude product is only about 15%. Although the efficiency of each step in the synthesis is 97% to 98%, the cumulative efficiency is not high. Take the chain lengths of 20mer and 50mer as an example, (97.5%) 20»60%, (97.5%) 50»28%. It can be seen that the purpose of the Oligo DNA content in the crude product is very low, even 10%. These impurities, especially the large amounts of salts and short chains present in the crude product, not only cause quantitative inaccuracy, but also affect the next reaction. Therefore, Oligo DNA must be purified.
Oligo DNA currently synthesized on a DNA synthesizer is mainly purified by polyacrylamide gel electrophoresis (PAGE). The purified product of this method has high purity and can be used in most molecular biology experiments. If cost savings are considered, for less demanding experiments, such as simple PCR reactions, desalting purification can be used.
Oligo DNA is measured by the OD260 value. In a 1 ml 1 cm path standard quartz cuvette, an Oligo solution with an absorbance of 1 at a wavelength of 260 nm was defined as 1 OD260. Although the base composition is not the same for each particular oligonucleotide, 1 OD260 Oligo DNA weighs approximately 33 mg and the average molecular weight per base is approximately 330 Da. Therefore, the mole number of Oligo DNA synthesized can be approximated by the following formula: mole number (mmol) = [OD value Ì33] / [base number Ì330] = 0.1 Ì [OD value / number of bases]