Sanger's DNA Squencing
1. Denaturation
The DNA is denatured at 94oc and prepared as single-stranded molecules and mixed with a short oligonucleotide complementary to the 3′ end of the DNA to be sequenced
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2. Primer attachment and extension of bases
The oligonucleotide acts as a primer for DNA synthesis catalyzed by DNA polymerase.
3. Oligonucleotide-primed material is split into four separate reaction tubes.
4 .Each tube receives a small amount of a [32P] labelled dNTP whose radioactivity allows the newly synthesized DNA to be visualized by autoradiography
5. Pairing
Each tube receives an excess of the four nonradioactive dNTP molecules and a small amount of one of the four ddNTPs. DNA polymerase is then added to the reaction mixture
6. Termination
As the polymerase replicates the DNA, it occasionally incorporates a ddATP residue instead of a dATP residue, and synthesis of the growing DNA chain is terminated
7. Production of newly synthesized DNA fragments of different lengths, each ending with A
8. Gel electrophoresis
The fragments are resolved by size. The sequence of the DNA molecule can then be read from an autoradiograph of the gel.
