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DNA-Sequencing

Explain the two main methods of sequencing, the dideoxy (Sanger) method and the basic method of 'pyrosequencing'

Mechanistic details for both Sanger and Pyrosequencing are provided.

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Question 1
Explain the two main methods of sequencing, the dideoxy (Sanger) method and the basic method of 'pyrosequencing'

The dideoxy method relies on properties of enzymes called DNA polymerases. These are enzymes that create new DNA polymers starting from individual nucleotides. However, for a DNA polymerase to work, it needs a "template" of single-stranded DNA on which to create the new polymer. DNA polymerase adds a new nucleotide to the 3′ end of a growing DNA chain, but the base of the new nucleotide must be able to base pair (ie, be complementary) to the base on the template over which the polymerase is positioned. After the addition of that nucleotide, the polymerase moves to the next nucleotide on the template, and adds a new nucleotide to the 3′ end of the growing chain. Again, the new nucleotide must be complementary to the next base in the template. When the process is completed, the DNA polymerase will have made a new DNA chain whose nucleotide sequence is completely complementary to the template DNA. For DNA sequencing using the chain-termination method (also termed Sanger or dideoxy method), four reactions are performed, each using theDNA to be analyzed as a template for a DNA polymerase reaction, and each containing one of the four dideoxynucleotides ...

Solution Summary

Here, two main methods of sequencing, the dideoxy (Sanger) method and the basic method of 'pyrosequencing' are explained, compared and contrasted.

Mechanistic details for both Sanger and Pyrosequencing are provided.

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