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First-generation sequencing utilized the method developed by Fredrick Sanger. It uses what is known as the dideoxy chain termination method.
DNA synthesis that uses the template (DNA that needs to be read via sequencing), primer (oligos), dNTPS (high energy nucleotides), and a polymerase enzyme. In addition to normal dNTPs, the dideoxy nucleotides are added in small amounts to each respective reaction. So, four different reactions having different dideoxy dNTPs are set up.
The equipment shown here is Life Technologies Gibco BRL Model SA Sequencing Gel Electrophoresis System.
As the DNA gets synthesized, dideoxy dNTPs get incorporated into it which leads to the termination of elongation. This causes generation of DNA fragments of varied length in the four reaction mixes. These reaction mixes are then run on an agarose gel of 60-70 cms in length in separate lanes. DNA molecules move as electric current is applied to the gel that is why it is known as electrophoresis. The sequence of the DNA is then determined by arranging the fragments on the gel linearly with respect to their lengths.
The equipment shown here is an ABI Prism 377 DNA Sequencer.
Human mitochondrial DNA (16,569 base pairs), and bacteriophage λ genome (48,502 base pairs) – the first complete genome - were sequenced using this method by Sanger.
Dr. 3730Seq also relies on Sanger sequencing technique, but uses capillary electrophoresis instead of the bulky agarose gels to resolve the DNA fragments.
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