DNA Sequencing

• The term DNA sequencing refers to methods for

determining the order of the nucleotides bases
adenine, guanine, cytosine and thymine in a
molecule of DNA.
Maxam-Gilbert sequencing
• A sequencing method based on a chemical degradation was
described by Maxam & Gilbert (1977).
• In this method, end-labelled DNA fragments are subjected to
random cleavage at adenine, cytosine, guanine, or thymine
positions using specific chemical agents (Table 2).
• The chemical attack is based on three steps :
• Base modification,
• Removal of the modified base from its sugar,
• And DNA strand breaking at that sugar positions.
• The products of these four reactions are then
separated using polyacrylamide gel electrophoresis.
• The sequence can be easily read from the four
parallel lanes in the sequencing gel.
• The template used in this sequencing method can be
either double-stranded (ds)DNA or ssDNA from
chromosomal DNA.
• In general, the fragments are first digested with an
appropriate restriction enzyme.
 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 is performed by chain

breakage at specific nucleotides.
 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′).

Sanger’s sequencing
 DNA sequencing by the Sanger method

The standard DNA sequencing technique is the Sanger method,

named for its developer, Frederick Sanger, who shared the 1980
Nobel Prize in Chemistry. This method begins with the use of
special enzymes to synthesize fragments of DNA that terminate
when a selected base appears in the stretch of DNA being
sequenced.
These fragments are then sorted according to size
by placing them in a slab of polymeric gel and applying an
electric field -- a technique called electrophoresis. Because of
DNA's negative charge, the fragments move across the gel toward
the positive electrode. The shorter the fragment, the faster it
moves. Typically, each of the terminating bases within the
collection of fragments is tagged with a radioactive probe for
identification.
 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′).

 Automated procedure for DNA
sequencing

A computer read-out of the gel generates a “false color” image

where each color corresponds to a base. Then the intensities are
translated into peaks that represent the sequence.
 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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