DNA sequencing- Maxam- Gilbert sequencing

By-
Dr. Dinesh C. Sharma
Head, Zoology
K.M. Govt. Girls P. G. College
Badalpur, G.B. Nagar
dr_dineshsharma@hotmail.com
“the process of determining the sequence of nucleotides (A, T, G, and C) in a piece of DNA”

Maxam-Gilbert sequencing published a DNA sequencing
method in 1977 based on chemical modification of DNA and
subsequent cleavage at specific bases. Also known as chemical
sequencing, this method allowed purified samples of double-
stranded DNA to be used without further cloning.
Maxam-Gilbert sequencing requires radioactive labeling at one 5' end
of the DNA and purification of the DNA fragment to be sequenced.
Chemical treatment then 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 concentration of the modifying chemicals is controlled
to introduce on average one modification per DNA molecule. Thus a
series of labeled fragments is generated, from the radiolabeled end to
the first "cut" site in each molecule. 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, yielding a series of dark bands each corresponding
to a radiolabeled DNA fragment, from which the sequence may be
inferred.

Maxam–Gilbert sequencing requires radioactive labeling at
one 5′ end of the DNA fragment to be sequenced (typically by
a kinase reaction using gamma-32P ATP) and purification of
the DNA.
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). For example, 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 concentration of the modifying chemicals is controlled to introduce on average
one modification per DNA molecule. Thus a series of labeled fragments is
generated, from the radiolabeled end to the first "cut" site in each molecule.
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, yielding a series of dark bands each showing the
location of identical radiolabeled DNA molecules. From presence and absence of
certain fragments the sequence may be inferred

https://www.youtube.com/watch?v=_B5Dj8PL4E0

5”- C T C G A G T G T A T C G
A C -3”
3”- T C T C T C A C A T A G C
T G -5”
| | | | | | | | | | | | | | |
1-Denturation @ 95O C
A C -3”
3”- T C T C T C A C A T A G C
T G -5”
Four samples (A+G), G, (T+C) and C
A+G G T+C C

1
A+G
2
G
3
T+C
4
C
A-Add radioactive phosphate (p32) in all four
2-Chemical Treatment
A C -3”
p32

1
A+G
2
G
3
T+C
4
C
B-Add Formic Acid in 1: Formic acid breaks link
between a purine (A+G) and the deoxyribose to which it attached
A C -3”
5”- C T
C
5”- C T C G A
G T
5”- C T C G
A
5”- C T C
G
5”- C T C G A G T
G T5”- C T C G A G T G T
A T C5”- C T C G A G T G T A T
7
Fragmen
t

1
A+G
2
G
3
T+C
4
C
C-Dimethyal Sulfate in 2: Methylation of
Guanine by DMS
A C -3”
5”- C T C
5”- C T C G A G T G T A
T C
5”- C T C G A G T
5”- C T C G A
4
Fragmen
t

1
A+G
2
G
3
T+C
4
C
D-Add Hydrazine in 3: The pyrimidines are
hydrolyzed by using hydrazine
A C -3”5”- C T C G A G T G T A T C G
A C5”- C T C G A G T G T A T C G
A5”- C T C G A G T G T A T
5”- C T C G A G T G
5”- C T C G A G
5”- C T
5”- C
8
Fragmen
t

1
A+G
2
G
3
T+C
4
C
E-Add Hydrazine and NaCl in 4: The addition of
NaCl to hydrazine reaction inhibits the reaction of thymine for –C
only reaction
A C -3”
5”- C
A
5”- C T C G A G T G T A T
5”- C T
4
Fragmen
t

1
A+G
2
G
3
T+C
4
C
F-Add Piperidine in all 4: The modified DNAs
cleaved by hot piperidine at the position of the modified base
A C -3”

3-Acrylamide Gel Electrophoresis
1
A+G
2
G
3
T+C
4
C
Anode
Cathode

• The negative charge of phosphate
backbone move the DNA
fragments towards the positively
charged anode
• Smaller DNA fragments migrate
more rapidly than larger DNA
fragments
Small
Large

5”- C T
C
5”- C T C G A G
T
5”- C T C G A
5”- C T C G
T
5”- C T C G A G T G T A T C
7

5”- C T C
T C
5”- C T C G A G T
5”- C T C G A
4

G A C
G A
5”- C T C G A G
5”- C T
5”- C
8

5”- C
C G A
5”- C T
4

5”- C T C
5”- C T C G A G T
5”- C T C G A
5”- C T C G
5”- C T C G A G T
G T
C
C G
5”- C T C
T C
5”- C T C G A G T
5”- C T C G A
G A C
G A
5”- C T C G A G
5”- C T
5”- C 5”- C
G A
5”- C T
C
C
C
C
T
T
T
T
G
G
G
G
A
A
A
C
A
G
C
T
A
T
G
T
G
A
G
C
T
C

https://www.khanacademy.org/science/high-school-biology/hs-molecular-
genetics/hs-biotechnology/a/dna-sequencing
https://www.youtube.com/watch?v=_B5Dj8PL4E0
https://en.wikipedia.org/wiki/DNA_sequencing

DNA sequencing- Maxam- Gilbert sequencing

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