Copying DNA the polymerase chain reaction

Of course, the product does not have a phosphate linker between the two nucleosides, and phosphorus is still in the wrong oxidation state. This is remedied by oxidation of the dinucleotide phosphite to a phos-photriester using iodine. We now have the required phosphate linker, though it is still protected with the cyanoethyl group. This is retained at this stage. 

The dimethoxytrityl ester protecting group is now removed by treatment with mild acid (CCI3CO2H), which is insufficiently reactive to hydrolyse the amide protection of bases, or the cyanoethyl protection of the phosphate. The coupling cycle can now be repeated using a phosphoramidite derivative of the next appropriate nucleoside. The sequences will be continued as necessary until the desired oligonucleotide is obtained. 

It then remains to remove protecting groups and release the product from the support. All of these tasks, except for the removal of the dimethoxytrityl group, are achieved by use of a single deprotection reagent, aqueous base (ammonia). The cyanoethyl groups are lost from the phosphates by base-catalysed elimination, and amide protection of the bases is removed by base-catalysed hydrolysis. The latter process also achieves hydrolysis of the succinate ester link to the support. 

The polymerase chain reaction (PCR), developed by Mullis, is a simple and most effective way of amplifying, i.e. producing multiple copies of, a DNA sequence. It finds applications in all sorts of areas not immediately associated with nucleic acid biochemistry, e.g. genetic screening, medical diagnostics, forensic science, and evolutionary biology. The general public is now well aware of the importance 

PCR makes use of the heat-stable enzyme DNA polymerase from the bacterium Thermus aquaticus and its ability to synthesize complementary strands of DNA when supplied with the necessary deoxyribonu-cleoside triphosphates. We have already looked at the chemistry of DNA replication (see Section 14.2.2), and this process is exactly the same, though it is carried out in the laboratory and has been automated. 

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