DNA Sequencing techniques explained

Let’s explore the different techniques of DNA sequencing, Sanger Sequencing versus Next Generation Sequencing (NGS), and see their pros and cons.

Sanger Sequencing

In the mid 70’s, Sanger developped a technique to sequence DNA, the Sanger Sequencing. With 99.99% accuracy it is nowadays considered the Gold Standard for clinical research. It works as follows:

The target sequence is first amplified by PCR. The temperature is increased until denaturation point where both strands of DNA separate. A primer is annealed at the 3' end of the DNA. The primed DNA is dispersed equally into four different reaction vessels. All vessel contain:

• DNA polymerases, for DNA extension
• Deoxyribonucleotide triphosohate (dNTP) including dATP, dCTP, dGTP, and dTTP, the building block of DNA

Then in each vessel, only one type of ddNTP is added, either ddATP or ddCTP or ddGTP or ddTTP. The polymerase synthetizes the complementary DNA strand (5' -> 3') with dNTP until it a ddNTP is used. The ddNTP, lacking a hydroxyl group at the 3' carbon, terminates the extension as is prevents the polymerases from attaching further dNTPs. As a result, each vessels end up with DNA fragments of different lengths all terminating with their respective ddNTP. Each ddNTP has a unique…