Sanger DNA Sequencing: Principle, Steps explained

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

How DiDeoxy nucleotide works in  Sanger DNA sequencing?

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 figure)

Sanger DNA sequencing steps and reading the gel

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.

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