Automated DNA Sequencing

Question 1

What are the steps involved in automated DNA sequencing?

Answer 1

• the primer anneals to the 3’ end of the template strand, allowing DNA polymerase to attach
• DNA polymerase adds free nucleotides according to base pairing rules so the strand grows
• if a modified nucleotide (terminator base) is added the polymerase enzyme is thrown off and the reaction stops on that template strand
• as the reaction proceeds many molecules of DNA are made and the fragments vary in size, the final added nucleotide of each strand is tagged with a specific colour
• the fragments are then run by electrophoresis to separate the fragments by size
• the strands are then run through a machine and a laser reads the sequence in order of size from the bottom to the top
• there is a sequence of colours displayed so the DNA sequence can be built up one base at a time

Question 2

What is included in the reaction mixture?

Answer 2

• DNA polymerase
• many copies of the single stranded DNA fragment
• free nucleotides
• primers
• fluorescently labelled modified nucleotide - if they are added to the growing chain the DNA polymerase is ‘thrown off’ and the strand cannot have any further nucleotides added to it, each different nucleotide has a different coloured fluorescent marker attached to it