Method of the Month — Sanger Sequencing
January 2022
Our next series of methods is going to shift the focus from gel electrophoresis to DNA sequencing. In this series, we’ll cover a range of techniques from the original method, Sanger sequencing, to next-generation sequencing.
Sanger sequencing, a technique that’s been around since the ’70s and known as the “chain termination method”, is a method that allows you to establish the nucleotide sequence of a DNA fragment. Sanger sequencing can be performed manually, but it is more commonly done in a sequencing machine, which is a lot quicker and much more efficient. There are three general steps to the process:
First, your original DNA sequence is used as a template for a particular PCR called chain-termination PCR. For a review of how PCR works, check out this article. Chain-termination is almost identical to regular PCR except that in the chain-termination method, you add modified nucleotides to the mix, called dideoxyribonucleotides (ddNTPs). These ddNTPs lack the 3’-OH group necessary to form the phosphodiester bond between nucleotides — in short, once a ddNTP is added to the growing strand, it halts the extension since another nucleotide cannot be added without that 3’-OH. Each ddNTP also has a specific fluorescent tag on it, one for As, one for Gs, one for Cs, and one for Ts.
Once the chain-termination process is complete, the second step in Sanger sequencing involves separating your oligonucleotide PCR products by size, which is done by gel electrophoresis. I’m sure a lot of you are familiar with this technique, but if not, you can check out our summary here. In the gel, the smaller oligonucleotides will travel farther, and the larger ones will end up closer to their starting point.
The final step in Sanger sequencing is simply reading the gel to determine the sequence of your original DNA fragment. Each ddNTP corresponds to a specific nucleotide in the original sequence, so reading the fragments from shortest to longest will give you the sequence of the DNA strand. For example, the shortest fragment must be terminated at the first nucleotide from the 5’ end (since DNA Polymerase synthesizes from 5’-3’), the second shortest fragment must be terminated at the second nucleotide from that 5’ end, and so on. In order to determine which fragments have been terminated by which ddNTPs, researchers use a laser to excite the nucleotides’ different fluorescent tags. This displays the fluorescent peaks of every ddNTP corresponding to the original nucleotides, making each fragment and related ddNTP easily identifiable.
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