Sanger Chain Termination Sequencing
What is DNA Sequencing?
Definition: DNA sequencing is a technique used to
determine the order of nucleotide sequence in a DNA molecule
In
DNA, the sugar-phosphate backbone remains the same whereas the order or bases
changes in different genes or DNA fragments. Therefore, DNA sequencing is
essentially the determination of order of base sequence in a DNA molecule
Sanger
Chain Termination method
Principle: Use of DiDeoxy nucleotides or ddNTPs as
chain terminators
The
process is based on the detection of labelled chain-terminating nucleotides
that are incorporated by a DNA polymerase during the synthesis of the template
strand
DNA
is made up of nucleotides. A nucleotide consists of a sugar, a nitrogenous base
and a phosphate.
In
a normal nucleotide or dNTP, the 3rd Carbon of deoxyribose sugar has
an OH group. This free 3’OH is needed
for ester bond formation by the incoming nucleotide during template strand
synthesis.
Watch our video Difference between deoxynucleotide and dideoxy nucleotide
How
Dideoxy nucleotide works in Sanger DNA sequencing?
In
the case of ddNTP, 3rd Carbon has H; without O or ddNTP lacks free 3
OH group. Whenever ddNTP is incorporated into a growing chain randomly, 3’H
cannot form ester bond with the next nucleotide. So, chain termination occurs.
That is why this method is also called as chain termination method.
Watch the above video for easy understanding
Steps
of Sanger DNA sequencing Procedure
Step
1 Preparation of 4 Reaction mixtures
Each
with ss template or unknown DNA, radioactively labelled primer, DNA polymerase
and 4 dNTPs (dATP, dCTP, dGTP and dTTP) and each reaction mixture with a
different ddNTP (ddATP for tube A, ddCTP for tube B, ddTTP for tube C, ddGTP for tube D)
Step
2 Chain Termination by ddNTP
Let
us take the case of tube A
Tube
A has ddATP, ddATP will be randomly incorporated into some of the newly synthesized
strands in the place of dATP. When ever ddATP is added, chain termination
occurs.
The
result is, in tube A differently sized DNA fragments are formed all ending in
ddATP or A (see figure)
The
same happens with other tubes, that leads to the formation of DNA fragments
ending in C, T and G respectively in tube B, C and D. (See the
Step
3: Gel electrophoresis
After
the completion of reaction, the products of 4 tubes are loaded separately to 4
lanes of a polyacrylamide gel and fragments are separated by size.
When
all the fragments of the tubes are separated by electrophoresis in four
different lanes, each DNA fragment differ from the next fragment by a single
base.
Step
4: Gel Analysis and Determination of DNA sequence
Autoradiography
is used to visualize radiolabeled DNA bands. All fragments in lane 1 ends in A,
lane 2 ends in C, lane 3 ends in T, lane 4 ends in G.
We
can read the gel from bottom to top to get the sequence of template strand.
From template strand sequence, we can deduce the sequence of unknown strand, because
of base pairing complementarity of DNA. This is the manual method Sanger DNA sequencing
method
The
process is based on the detection of labelled chain-terminating nucleotides
that are incorporated by a DNA polymerase during the synthesis of the template
strand.
Try this Quiz on DNA sequencing
What
is Automated Sanger DNA sequencing?
In
automated Sanger sequencing, different fluorescent labels are used for
different ddNTPs.
Aa computer reads each band of the capillary gel, in accordance with the fluorescence to identify each terminal ddNTP. The output shows fluorescent peak of each nucleotide along the length of the template DNA called a chromatogram.
