Question

Explain the Sanger sequencing method (p. 221 - 222) (Tablet p.249 – 250)
i. In your answer refer to the primer, type of nucleotides and polymerase
c. Read a DNA sequence from an autoradiograph (p. 221 Fig 8.16) (Tablet p.249)

Answer 1

Sanger sequencing is a DNA sequencing method that uses ddNTP. This analogue of ddNTP lack 3'OH because of which the polymerization of nucleotides into a DNA strand. The polymerization of nucleotides into a DNA strand as it happens in a reaction with polymerase in DNA replication occurs through a phosphodiester bond. A phosphodiester bond is formed between a 3'OH and 5' PO4 in which water leaves. In case of Sanger's method, as ddntp lack 3',OH, the replication stops .4 different reaction mixtures are taken in which a particular ddNTP of a particular nucleotide a or t or g or c are added. These are then separated on the gel electrophoresis and the sequence is read to deduce the sequence of the gene that was replicated.

A T G C

_

_.  

_ .

_

_

For example assume that the strands have separated as above.

Read them from below, 5' CGATA.'3

Which means 3' G CTAT '5 is the sequence of the DNA that had to be sequenced originally.

The method uses ddNTP, dNTP, dna polymerase