method of DNA sequencing

Last reviewed 01/2018

Manual method:

• a fragment of double-stranded DNA whose sequence in unknown is cloned into a plasmid vector. Heat is applied to separate the two strands. A primer is added which then hybridizes to one of the two template strands. The DNA primer has a radioactive tag. The plasmid-primer hybrid is then added to four tubes. Each tube contains DNA polymerase, all four nucleotides, and a single dideoxynucleotide (each tube contains a different dideoxynucleotide). The addition of the DNA polymerase extends the DNA primer which incorporates nucleotides, and occasionally dideoxynucleotides into growing DNA chains. The incorporation of the dideoxynucleotide prevents elongation of the DNA chain. The newly formed DNA strands are then analysed. The strands of DNA are loaded onto a special sequencing gel that allows the separation of DNA strands differing by only one base. Electrophoresis then distributes the strands according to size with the smallest ones traveling the farthest along the gel. The length of the strand is important because it means that the shortest strands have incorporated the nucleotides closest to the primer. The sequence of the DNA from 5' to 3' can be deduced from reading the sequencing pattern from the bottom of the gel up.

Automated method:

• this is similar to the manual method but instead uses a DNA primer labelled with four different fluorescent tags. The process of deduction of the nucleotide sequence relies on the emission of a specific fluorescent signal of each fragment. The wavelength of each signal corresponds to a dideoxynucleotide incorporated in the elongation reaction. Computerised analysis translates the signals as a nucleotide sequence.