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DNA Sequencing Methodology: Dideoxy and Pyrosequencing

I have two questions in biology and I need perfect answer
Question 1
Explain the two main methods of sequencing, the dideoxy (Sanger) method and the basic method of 'pyrosequencing'
Question 2
Show, with examples, how the 'pyrosequencing' method has been useful for very high throughput applications
thank you

Solution Preview

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 (dideoxyadenosine triphosphate [ddA], dideoxycytidine triphosphate [ddC], dideoxyguanosine triphosphate [ddG], and dideoxythymidine triphosphate [ddT]). In each reaction, chain elongation will terminate when the dideoxynucleotide is incorporated at the position of its complementary nucleotide in the template. This will result in a ...

Solution Summary

Here, the Sanger (dideoxy) and the pyrosequencing, DNA sequencing methods are described in detail and example for high throughput pyrosequencing applications are provided.

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