DNA sequencing is the process of working out
the order of the building blocks, or bases, in a strand of DNA.
Before we can sequence the DNA, it has to be cut up into smaller pieces
that are inserted into plasmid DNA
and then put into bacterial cells.
This makes it possible to produce lots and lots of copies of it
as the bacterial cells multiply.
The DNA is then isolated from the bacteria and sent for sequencing.
The isolated DNA is transferred to a plate where the sequencing reaction will take place.
A mixture of ingredients is added.
These include free DNA bases (A, C, G, T),
DNA polymerase enzyme
and DNA primers.
Modified DNA bases labelled with coloured, fluorescent tags are also added.
These are called terminator bases.
To start the sequencing reaction, everything is heated to 96 degrees Celsius.
This separates the DNA into two single strands.
The temperature is then lowered to 50 degrees.
This enables the DNA primers to bind to the plasmid DNA.
The temperature is then increased to 60 degrees
and the enzyme DNA polymerase binds to the primer DNA.
DNA polymerase starts making a new strand of DNA
by adding unlabelled DNA bases to the target DNA.
It continues to add DNA bases until a terminator base is added.
These terminator bases have been chemically altered
so that no more bases can be added to the new strand of DNA.
Once a terminator base is added, the DNA polymerase enzyme
stops making DNA and falls away from the strand.
Everything is then heated to 96 degrees Celsius again
to separate the new DNA strand from the original strand.
This process of heating and cooling is repeated again and again
to produce lots of fragments of DNA of different lengths.
The length of each fragment depends on when a terminator base got added.
To read the sequence of the DNA the various fragments are separated by length
using a process called electrophoresis.
A capillary tube is lowered into each well of the plate and an electrical charge is applied.
This causes the negatively-charged DNA molecules to move through the capillary tube.
Each capillary contains a porous gel.
The shorter fragments of DNA move through the gel more easily than the longer DNA fragments.
As a result, the fragments become arranged by size, from the shortest to the longest.
As the DNA fragments come to the end of the capillary,
a laser makes the terminator bases light up.
The colour is detected by a camera and recorded.
Each terminator base is labelled with a different colour.
‘A’ fluoresces green, ‘C’ blue, ‘G’ yellow, and ‘T’ red.
The shortest DNA fragments will be read first, and the longest read last.
The sequencing machine records the colour of the terminator bases as a series of coloured blocks.
Each coloured block represents the labelled, terminator base at the end of each fragment of DNA.
By converting the colours into letters we get the sequence of our piece of DNA.