What Is Sanger Sequencing Method

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∟What Is Sanger Sequencing Method

Provides a quick introduction of PCR (Polymerase Chain Reaction, which is a chemical synthesis process developed by Kary Mullis to rapidly amplify sequences of a DNA.

Now let's go back to Sanger Sequencing method.

What Is Sanger Sequencing Method? - Sanger Sequencing is based on the chain-termination method developed by Frederick Sanger to determine the sequence of nucleotide bases in a given DNA.

Main steps of Sanger Sequencing Method:

1. PCR Amplification - The standard PCR process is used to replicate the target DNA in a large amount.

2. PCR Clean-Up - This is to remove unbound primers, unused dNTPs (dATP, dCTP, dGTP, and dTTP), unused DNA templates, and other unused components.

3. Cycle Sequencing - This is a modified PCR called Chain-Termination PCR, which generate DNA fragments that end at each every nucleotide base of the target DNA sequence.

4. Sequencing Clean-Up - This is to remove unbound primers, unused dNTPs and ddNTPs, unused DNA templates, and other unused components.

5. Capillary Electrophoresis - This is to sort DNA fragments generated from the previous step by the length, and read them sequentially from the shortest to the longest.

6. Data Analysis - The DNA sequence identified from the previous step is used to compare with the reference DNA sequence to find any mutations.

The picture (source: thermofisher.com) below illustrates the above steps:

The key step of the Sanger Sequencing Method is Cycle Sequencing, also called Chain-Termination PCR. It can be described below in details:

1. Chain-Termination PCR: Preparation -

Take 4 separate tubes labeled A, C, G and T.
Divid the large amount of DNAs (amplified from the single target DNA) into 4 portions and add them to 4 tubes.
Add dNTPs (dATP, dCTP, dGTP, and dTTP) to each tube, as in the PCR process.
Add ddNTPs: ddATP to Tube A, ddCTP to Tube C, ddGTP to Tube G, and ddTTP to Tube T.
Add primers to each tube. They are radioactively labeled differently for different tubes.

2. Chain-Termination PCR: Synthesis -

The Synthesis of new DNA strands starts with primers in each tube.
A dNTP or ddNTP is added to a new DNA strand to extend the strand.
If a dNTP is added to the new DNA strand, step 2 is repeated.
If a ddNTP is added to the new DNA strand, the new extension process is terminated.

At the end of the synthesis step:

Tube A contains a mixture of new DNA strand fragments ending with nucleotide A with different lengths.
Tube C contains a mixture of new DNA strand fragments ending with nucleotide C with different lengths.
Tube G contains a mixture of new DNA strand fragments ending with nucleotide G with different lengths.
Tube T contains a mixture of new DNA strand fragments ending with nucleotide T with different lengths.

In theory, if we add enough amplified DNAs, primers, dNTPs, and ddNTPs, with a proper balance, we will have new DNA strand fragments end at each and every nucleotide base in the original target DNA amount 4 tubes.

After purification of DNA strands fragments collected from the Chain-Termination PCR process, they will be separated, sorted and red by electrophoresis on a plyacrylamide gel.

The plyacrylamide gel has 4 lanes, one for each tube. Driven by an electric current, DNA strand fragments from tubes will through the gel. But shorter and lighter fragments move faster than longer and heavier fragments. This allows us to read one fragment at a time from the shortest one to the longest one.