Sanger method
The sequencing reaction
DNA sequencing involves making copies of the original DNA sequence; the reaction uses the building blocks of DNA and an enzyme called DNA polymerase to add new bases to a growing DNA chain. However, this reaction is set up so that it doesn't work all the time - we don't want a perfect copy of our DNA.
First, the two strands of the DNA in the pUC clones are separated. Then, a short piece of DNA called a primer is added. The primer will bind specifically to a DNA sequence in the pUC molecule. It also serves as a starting point for building a new DNA chain. New building blocks of the DNA chain are added together with DNA polymerase. If this were all, the reaction would copy a new chain until it stopped.
The key to all DNA sequencing and the development that gave Fred Sanger his second Nobel Prize was the dideoxy base. DNA is called deoxyribonucleic acid because the ribose sugar part of the molecule is lacking an oxygen atom found in normal ribose. Dideoxy bases lack a second oxygen atom that is required to extend the growing DNA chain. This means that when a dideoxy base is incorporated into a DNA molecule, the chain stops or terminates.
The reactions are set up so that there is a mix of 'normal' and dideoxy bases. At any position, either a normal base will be added, so the chain can continue to grow, or a dideoxy base will be added, so the chain terminates. After many cycles of copying, all the possible chain-termination molecules are produced: the reaction has stopped at every base.
