Sanger Sequencing

Sanger Sequencing is one of the oldest and most reliable DNA sequencing methods. It is a laboratory technique that allows one to determine the exact order of the bases in a DNA fragment or sequence. It was invented by Frederick Sanger in 1977 and continues to be one of the most widely used techniques for DNA sequencing and gene prediction.

Capillary Electrophoresis for DNA Fragment Analysis

Capillary electrophoresis is a technique to separate DNA fragments based on their size. The capillary tube is filled with a buffer solution that contains a dye that binds to the DNA fragments. 

 

Capillary Electrophoresis -Sanger Sequencing Procedure

The Sanger sequencing reaction mixture is prepared by adding 10 μl of the DNA sample, 2 μl of polymerase (10 units), 4 μl of deionized water and 1U Taq DNA polymerase. The entire mixture is then incubated at 50°C for 3 hours followed by a cooling step to room temperature for 30 minutes before it is ready for electrophoresis. The DNA fragments are separated on an agarose gel using buffer A (10 mM Tris-HCl pH 8.0) and buffer B (50 mM Tris-HCl pH8). The gel will have bands corresponding to each nucleotide base sequence in your sample if they were accurately sequenced; these bands are used as markers throughout this procedure.

Automated Sanger Sequencing Reaction Set-Up

The steps of automated Sanger sequencing are as follows:

• Make up a reaction mixture containing 100 uL of 3' terminal nucleotides and 20 uL of 5' terminal primer (10 mM Tris-HCl pH 8.3; 2mM MgCl2; 1 mM DTT).
• Add 50 ng DNA template, 0.5 uL dNTPs, 1 μM dNTP mix (100 mM each), 0.2 μM primer mix (10 mM each) and 40 μL nuclease-free water to your reaction vessel (50x volume).
• Incubate at 37°C for at most 30 minutes,

Molecular biologists use the Sanger sequencing technique to determine the base-by-base ordering of DNA fragments. The method has developed into a powerful tool for investigating all genetic changes.