DNA SEQUENCING

Determining the order of bases in a section of DNA

• To analyze gene structure and its relation to gene
expression as well as protein conformation
PURPOSE

 Code of life (human genome)

• Detecting mutations
• Typing microorganisms
• Identifying polymorphisms
DNA SEQUENCING METHODS
1. Sanger method Modern sequencing equipment uses
the principles of the Sanger technique.
2. Second generation sequencing method
Dideoxy (Sanger) method
4 steps:
Denaturation
Primer attachment and extension of bases
 Termination
Gel electrophoresis
Dideoxy (Sanger) method
ddNTP 2,
The 3︠ dideoxynuleotide
No 3︠ hydroxyl group only hydrogen atom
Terminates chain when incorporated
Add enough so each ddNTP is random and completely
incorporated at each bases
Dideoxynucleotides
We run the reactions, however, in the presence of a
dideoxyribonucleotide. This is just like regular DNA,
except it has no 3' hydroxyl group- only hydrogen-
once it's added to the end of a DNA strand, there's no
way to continue elongating it.
Replicating a DNA strand in the presence of
colored didNTPs

Most of the time when a dNTPs is required to make

the new strand, the enzyme will get a good one and
there's no problem. However, 5% of the time, the
enzyme will get a didNTPs (hydrogen only) and that
strand can never again be elongated. Sooner or later all
of the copies will get terminated by a didNTPs colored
Putting all four deoxynucleotides into tube for
termination :

Then we ran the reaction with all four of the dideoxy

nucleotides (A, G, C and T) present, and with different
fluorescent colors yellow green red blue on each.
know the color codes ... just read the colors from
bottom to top: TGCGTCCA-(etc).
That's exactly what we do to sequence DNA, then - we
run DNA replication reactions in a test tube, but in the
presence of trace amounts of all four of the dideoxy
terminator nucleotides. Electrophoresis is used to
separate the resulting fragments by size and we can
'read' the sequence from it, as the colors march past in
order.
Dideoxy method
Run four separate reaction each with different
ddNTPS
Run on a gel four separate lanes
Read the gel from the bottom up
So what’s wrong with it?
The dideoxy method is good only for 500-750 bp
reactions
Expensive
Takes a while
The human genome is about 3 billion bp
Pyrosequencing method
1st Method
Solid phase
 Immobilized DNA
 3 enzymes
 Wash step to remove nucleotides after each addition
Pyrosequencing method
2nd method
Liquid phase
3 enzymes + apyrase (nucleotide degradation enzyme)
Summary
DNA sequencing is a common procedure
Dideoxy method
 Chain termination method
 Best for small DNA segments
Pyrosequencing
 Sequence by synthesis
 Accurate and fast