DNA Sequencing

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 Objectives
 Compare and contrast the chemical (Maxam/Gilbert) and
chain termination (Sanger) sequencing methods.
 List the components and molecular reactions that occur in
chain termination sequencing.
 Discuss the advantages of dye primer and dye terminator
sequencing.
 Describe examples of alternative sequencing methods, such
as bisulfite sequencing and pyrosequencing.

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 Sequencing Methods

Maxam/Gilbert chemical sequencing

Sanger chain termination sequencing
Pyrosequencing
Array sequencing

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 MaXam Gilbert chemical
sequencing
 For Maxam-Gilbert sequencing a technique called end labeling
is used, in which a radioactive atom is added to the ends of the
DNA fragment being sequenced.

 The first step in this process is to use an enzyme, called a

restriction endonuclease, to cut the DNA at a specific sequence.

 If the restriction endonuclease is Hind III, for example, the

sequence AAGCTT will be cut.

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 Maxam ..
 The ends of the DNA segment made by the restriction
endonuclease will have phosphate groups (-PO32-) at its ends.

 An enzyme called a phosphatase is used to remove the

phosphate group.

 Another enzyme, called a kinase, is then used to add a

radioactive phosphate in its place.

 This reaction will add radioactive atoms onto both ends of

the DNA restriction fragment

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 Maxam
 A second restriction endonuclease is used to make a cut within
the end-labeled fragment and gele electrophoresis is then
employed to separate the two resulting subfragments from each
other.

 Each subfragment now has one labeled and one unlabeled end.

 The subfragment whose sequence is to be determined is cut out

of the gel to purify it away from the other end-labeled
subfragment.

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 Maxam…
 The end-labeled piece of DNA is then divided and the fragments are
placed in four separate tubes.

 They are then treated with different chemicals that weaken and break the
bond holding the base to the backbone of the DNA molecule.

 These chemicals are base specific. In other words, one chemical causes
the "C" reaction, in which the bond holding the C base in position is
broken.

 Another chemical breaks the bond holding the G in place (the "G"
reaction).

 Another breaks both G and A bases from the DNA backbone

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  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  are methylated by dimethyl sulfate, and the
pyrimidines (C+T) are hydrolysed using hydrazine.

 The addition of salt (sadium 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.

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 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

DMS=Dimethyl sulphate, FA= formic acid

H=Hyderazide, S=salt

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 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′).

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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

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 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.

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 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

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 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.
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 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.

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 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′).

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 Cycle Sequencing

Cycle sequencing is chain termination sequencing

performed in a thermal cycler.

Cycle sequencing requires a heat-stable DNA

polymerase.

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 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.

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 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

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

ddA
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 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
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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

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Dye Terminator Sequencing
The DNA ladder is read on an electropherogram.

Slab gel Capillary

Electropherogram

5′ AGTCTG
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 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

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 Alternative Sequencing Methods:
Pyrosequencing
Pyrosequencing is based on the generation of
light signal through release of pyrophosphate
(PPi) on nucleotide addition.
DNAn + dNTP  DNAn+1 + PPI

PPi is used to generate ATP from adenosine

phosphosulfate (APS).
APS + PPI  ATP

ATP and luciferase generate light by

conversion of luciferin to oxyluciferin.

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 Alternative Sequencing Methods:
Pyrosequencing
Each nucleotide is added in turn.

Only one of four will generate a light signal.

The remaining nucleotides are removed enzymatically.

The light signal is recorded on a pyrogram.
DNA sequence: A T C A GG CC T

Nucleotide added : A T C A G C T
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 Alternative Sequencing Methods:
Bisulfite Sequencing
Bisulfite sequencing is used to detect methylation in
DNA.

Bisulfite deaminates cytosine, making uracil.

Methylated cytosine is not changed by bisulfite

treatment.

The bisulfite-treated template is then sequenced.

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 Alternative Sequencing Methods:
Bisulfite Sequencing
The sequence of treated and untreated templates is
compared.

Methylated sequence: GTC Me GGCMe GATCTATC Me GTGCA…

Treated sequence: GTC Me GGCMe GATUTATC Me GTGUA…

DNA Sequence:

(Untreated) reference: ...GTCGGCGATCTATCGTGCA…

Treated sequence: ...GTCGGCGATUTATCGTGUA…

This sequence indicates that these Cs are methylated.

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 Summary
 Genetic information is stored in the order or sequence of
nucleotides in DNA.

 Chain termination sequencing is the standard method for the

determination of nucleotide sequence.

 Dideoxy-chain termination sequencing has been facilitated

by the development of cycle sequencing and the use of
fluorescent dye detection.
 Alternative methods are used for special applications, such as
pyrosequencing (for resequencing and polymorphism
detection) or bisulfite sequencing (to analyze methylated
DNA).
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