DNA Sequencing

Imran Amin
Sequencing Methods
• Maxam/Gilbert chemical sequencing
• Sanger chain termination sequencing
Maxam–Gilbert sequencing

Maxam–Gilbert sequencing is a method of DNA sequencing developed by Allan

Maxam and Walter Gilbert in 1976–1977. This method is based on nucleobase-
specific partial chemical modification of DNA and subsequent cleavage of the DNA
backbone at sites adjacent to the modified nucleotides
 Chemical treatment generates breaks at a small proportion of one or two of the
four nucleotide bases in each of four reactions (G, A+G, C, C+T).

 The purines (A+G) are depurinated using formic acid, the guanines (and to some
extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines
(C+T) are hydrolysed using hydrazine. The addition of salt (sodium chloride) to
the hydrazine reaction inhibits the reaction of thymine for the C-only reaction

 The modified DNAs may then be cleaved by hot piperidine;(CH2)5NH at the

position of the modified base

 The fragments in the four reactions are electrophoresed side by side in

denaturing acrylamide gels for size separation. To visualize the fragments, the gel
is exposed to X-ray film for autoradiography. From presence and absence of
certain fragments the sequence may be inferred
Maxam-Gilbert Sequencing

DMS FA H H+S

G G C C
G A T C
G G T
G G C C
C
A T
G C
A C
A T
Maxam-Gilbert Sequencing

3′
A
A
G
G G+A T+C C
C
Longer fragments A
A A
C
G
T
G
Shortest fragments C
G
A
G
5′

Sequencing gels are read from bottom to top (5′ to 3′).

A sequencing gel
Chain Termination (Sanger) Sequencing

• A modified DNA replication

reaction.
• Growing chains are terminated
by dideoxynucleotides.
Chain Termination (Sanger) Sequencing
The 3′-OH group necessary for formation of the phosphodiester bond
is missing in ddNTPs.

Chain terminates
at ddG
Chain Termination (Sanger) Sequencing
• A sequencing reaction mix includes labeled primer and template.

Primer

5′OP- -3′ OH
TCGACGGGC…
Template
Template area to be sequenced

• Dideoxynucleotides are added separately to each of the four tubes.

Chain Termination (Sanger) Sequencing

ddATP + ddA
A
four dNTPs dAdGdCdTdGdCdCdCdG

ddCTP + dAdGddC
C
four dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdCddC

ddGTP + dAddG
G four dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCddG

T
ddTTP + dAdGdCddT
four dNTPs dAdGdCdTdGdCdCdCdG
Chain Termination (Sanger) Sequencing
• With addition of enzyme (DNA polymerase), the primer is extended
until a ddNTP is encountered.
• The chain will end with the incorporation of the ddNTP.
• With the proper dNTP:ddNTP ratio, the chain will terminate
throughout the length of the template.
• All terminated chains will end in the ddNTP added to that reaction.
Chain Termination (Sanger) Sequencing
• The collection of fragments is a sequencing ladder.
• The resulting terminated chains are resolved by electrophoresis.
• Fragments from each of the four tubes are placed in four separate gel
lanes.
Chain Termination (Sanger) Sequencing
3′
G
G A T C G
Longer fragments T
A
ddG
A
A
T
C
Shorter fragments A
ddG T
G
5′

Sequencing gels are read from bottom to top (5′ to 3′).

Cycle Sequencing

• Cycle sequencing is chain termination sequencing

performed in a thermal cycler.
• Cycle sequencing requires a heat-stable DNA
polymerase.
Fluorescent Dyes
• Fluorescent dyes are multicyclic molecules that absorb and emit
fluorescent light at specific wavelengths.
• Examples are fluorescein and rhodamine derivatives.
• For sequencing applications, these molecules can be covalently
attached to nucleotides.
Fluorescent Dyes
• In dye primer sequencing, the primer contains fluorescent dye–
conjugated nucleotides, labeling the sequencing ladder at the 5′ ends
of the chains.

• In dye terminator sequencing, the fluorescent dye molecules are

covalently attached to the dideoxynucleotides, labeling the
sequencing ladder at the 3′ ends of the chains.

ddA

ddA
Dye Terminator Sequencing
• A distinct dye or “color” is used for each of the four ddNTP.
• Since the terminating nucleotides can be distinguished by color, all
four reactions can be performed in a single tube.

T The fragments are

AC distinguished by size and
GT
G “color.”

T
 Dye Terminator Sequencing

The DNA ladder is resolved in one gel lane or in a capillary.

GA
G A T C TC

G
T
C
T
G
A

Slab gel Capillary

 Dye Terminator Sequencing

• The DNA ladder is read on an electropherogram.

Slab gel Capillary

Electropherogram

5′ AGTCTG
Automated Sequencing
• Dye primer or dye terminator sequencing on capillary instruments.
• Sequence analysis software provides analyzed sequence in text and
electropherogram form.
• Peak patterns reflect mutations or sequence changes.

T/T T/A A/A

5′ AGTCTG 5′ AG(T/A)CTG 5′ AGACTG

Automated procedure for DNA
sequencing