Gene Sequencing

Gene Sequencing

• DNA sequencing: technique that allows genes to be isolated and read

• The sequencing reaction can only work on short lengths of DNA.

• We can use PCR or insert genes into bacteria to amplify the DNA we want to work with.

• Therefore the genome must first be broken up into sections using restriction enzymes, each section is sequenced individually and then assembled in the correct order.

• Fragments are then separated using electrophoresis (remember, separation is based on SIZE).

• Each fragment is then sequenced using an automated sequencer.

What do we need in the reaction mixture?

1. DNA polymerase

2. Many copies of the single strand of DNA to be sequenced

3. Primers 

4. Free DNA nucleotides, each type fluorescently labelled with a colour (terminator bases)

Method:

• The primer joins which allows DNA polymerase to attach

• DNA polymerase adds the free DNA nucleotides, using the complementary base-pairing rules, so the strand grows

• If a modified terminator nucleotide is added, the chain stops growing

• Therefore, whenever the terminator nucleotides are added, copying stops

• As there are many many (many) free nucleotides and template strands, the result is many different chains, all different lengths, each chain ending…