DNA Sequencing

We will discuss one method of reading the sequence of DNA. This method, developed by Sanger won him a second Nobel prize. To sequence a single stranded piece of DNA, the complementary strand is synthesized. Four different reaction mixtures are set up. Each contain all 4 radioactive deoxynucleotides (dATP, dCTP, dGTP, dTTP) required for the reaction and DNA polymerase. In addition, dideoxyATP (ddATP) is added to one reaction tube The dATP and ddATP attach randomly to the growing 3' end of the complementary stranded. If ddATP is added no further nucleotides can be added after since its 3' end has an H and not a OH. That's why they call it dideoxy. The new chain is terminated.. If dATP is added, the chain will continue to grow until another A needs to be added. Hence a whole series of discreet fragments of DNA chains will be made, all terminated when ddATP was added. The same scenario occurs for the other 3 tubes, which contain dCTP and ddCTP, dTTP and ddTTP, and dGTP and ddGTP respectively. All the fragments made in each tube will be placed in separate lanes for electrophoresis, where the fragments will separate by size.