Sanger sequencing was developed by Frederick Sanger and his colleagues in 1977. The development of this technique won Sanger the Nobel Prize in Chemistry in 1980.
From the 80's to the mid-2000's, Sanger sequencing dominated the DNA sequencing platform, bringing successful completion of the Human Genome Project (HGP) in 2003. Although this technique has been replaced by next generation sequencing methods, it is still used today for smaller-scale projects.
What is dideoxynucleotide?
A dideoxynucleotide (ddNTP) is an artifical molecule that lacks a hydroxyl group at both the 2' and 3' carbons of the sugar moiety. Compare this to a regular deoxynucleotide triphosphate (dNTP), which has the hydroxyl group on the 3' sugar.
The main purpose of the 3'-OH group is that it is used to form a phosphodiester bond between two nucleotides - this is what allows for a DNA strand to elongate.
DNA elongation cannot occur with ddNTPs
During DNA replication, an incoming nucleoside triphosphate is linked by its 5' α-phosphate group to the 3' hydroxyl group of the last nucleotide of the growing chain. With ddNTP, where there is no 3' hydroxyl group, this reaction cannot take place, so elongation is terminated.
Here is an image of how DNA elongation regularly occurs (with dNTP instead of ddNTP).
Now that we have seen the chemistry behind the ddNTP, let's look at how Sanger Sequencing works.
There are three main steps in Sanger Sequencing, as outlined below.
1) Clone DNA strands into a vector
The first step is to fragment the DNA and clone the fragments into vectors.
2) Attach primer
The second step is to anneal a synthetic oligonucleotide with length 17 to 24-mer. (An oligonucleotide is just a fancy name for a short strand of DNA). The oligonucleotide acts as a binding site for a primer and provides a 3' hydroxyl group, which is necessary to initiate DNA synthesis.
In order to recognize the sequence and identify precisely the first nucleotide of the target DNA, the primer is usually positioned 10 to 20 nucleotides away from the target DNA.
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