Dideoxy DNA Sequencing - Sanger method

Sanger sequencing is based on chain termination method using dideoxynucleotide. It was developed by Frederick Sanger and his collegues in 1977. It employs in vitro replication of DNA using Klenow fragments and necessary dNTPs with required concentration (very small as compared to dNTP) of ddNTPs. Here, the DNA fragment is denatured to its single strand and a primer (oligonucleotide of known sequence) is synthesized and added.
There are copies of multiple fragments in the sample. The ssDNA is added to 4 different tubes which consist of ddATP, ddGTP, ddCTP, and ddTTP dideoxynucleotides respectively. All the tubes consist of necessary ingredients for the replication; i.e. DNA polymerase (Klenow fragment), dNTPs, enzyme regulators (Mg2+ ions), etc. and are available collectively in form of Mastermix.
The primer added is radiolabelled so that after the PAGE the bands can be visualized by autoradiography.
To understand the process easily, please watch this video.
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