Sanger sequencing is a method for determining the sequence of nucleotide bases in a string of DNA. Fredrick Sanger developed it in 1977. This method is also known as the chain termination method.
This method was initially used for validating the DNA strings sequenced by third-generation techniques and eventually the entire chromosome.
Sanger sequencing works by following these three steps:
DNA sequencing for chain termination PCR
Size separation by gel electrophoresis
Gel analysis and determination of DNA sequence
Initially, a DNA primer that is complimentary to the DNA to the DNA to be sequenced is added. This acts as a starting point for the synthesis of the DNA. Under the effect of the four deoxynucleotides triphosphates (dNTP where N can be A, G, C, and T), the polymerase extends the DNA primer due to the complimentary dNTP till the template DNA strand.
Four dideoxynucleotide triphosphates (ddNTP) where N can be A. G, C, and T) are labeled to figure out which nucleotide is incorporated in the chain of nucleotides with a fluorescent dye used to terminate the synthesis reaction.
Compared to the dNTPs, the ddNTPs have an oxygen atom removed from the ribonucleic part, hence why it can not link with the successive nucleotide. This step gives us chain terminated oligonucleotides.
After the synthesis, the products of the reaction are loaded into four columns of a single gel depending on the diverse chain terminating nucleotide and are subjected to gel electrophoresis.
In gel electrophoresis, a current is applied to the DNA samples. Since DNA is negatively charged, the oligonucleotides are pulled towards the positive electrodes on the opposite side of the gel. Their size determines the speed at which the oligonucleotides move. Thus, the oligonucleotides are arranged from the smallest to the largest size while reading the gel from the bottom to the top.
This step involves the gel's reading process to determine the DNA sequence. Each terminal ddNTP corresponds to a specific nucleotide in the sequence. The DNA polymerase synthesizes the DNA in the 5' to 3' direction, starting from the primer DNA. Each ddNTP is separated based on the fluorescence they excite, resulting in a light emittance.
A chromatogram is used to show the fluorescent peak of each nucleotide as well as its length. Thus, by reading the gel bands from the smallest to the largest, we determine the sequence of the template DNA.
Sanger sequencing and PCR use similar starting materials and can be used together. But neither of these can replace the other one.
The purpose of PCR is to amplify the DNA strands, variable length fragments of the DNA may be produced by accident, but the purpose is to duplicate the DNA sequence.
On the other hand, Sanger sequencing aims to produce all possible length fragments of the DNA up to the entire length of the template DNA. This is why ddNTPs are used along with PCR in this method.
PCR produces many copies of the template DNA. Having more than one template DNA makes Sanger sequencing efficient.
The commercial availability of the next-generation sequencing technologies has succeeded Sanger sequencing. This sequencing method remains widely used because it is cost-efficient and highly accurate.
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