DNA Sequencing
Sequencing means finding the order of nucleotides
on a piece of DNA .
Nucleotide order determines amino acid order, and
by extension, protein structure and function
(proteomics) .
An alteration in a DNA sequence can lead to an
altered or non functional protein, and hence to a
genetic disorder .
DNA sequence is important to detect the type of
mutations in genetic diseases and offer hope for the
eventual development of treatment DNA .
Methods of sequencing
there are two main methods of DNA
sequencing:
1-Sanger dideoxy (primer extension/chain-termination)
method: most popular protocol for sequencing, very
adaptable, scalable to large sequencing projects
2-Maxam-Gilbert chemical cleavage method: DNA is
labelled and then chemically cleaved in a sequence-
dependent manner. This method is not easily scaled
and is rather tedious .
Modern sequencing equipment uses the principles of
the Sanger technique.
History of Sequencing
”Sanger Sequencing” developed by Fred Sanger et al in the
mid 1970’s
Uses dideoxynucleotides for ”chain termination”,
generating fragments of different lengths ending in ddATP,
ddGTP, ddCTP or ddTTP.
The 3’ hydroxyl has been changed to a hydrogen in ddNTP’s, which
terminates a DNA chain because a phosphodiester bond cannot form
at this 3’ location
The Sanger Technique Principle ::
The Sanger Technique uses dideoxynucleotides (dideoxyadenine,
dideoxyguanine, etc) These are molecules that resemble normal nucleotides
but lack the normal -OH group.
Because they lack the -OH (which allows nucleotides to join a growing DNA
strand), replication stops.
Requirements for Sanger Method
DNA to be sequenced must be in single strand form.
The region to be sequenced must be 3’ flanked by known
sequence.
Reagents needed are:
• A primer complementary to the known region to start and direct
chain synthesis. (15-30 nucleotides in length)
• DNA polymerase.
• 4 deoxynucleotide triphosphates (dNTPs).
• 4 dideoxynucleotide triphosphates (ddNTPs) ( small proportion ) .
Mechanism of DNA polymerization
O
O
5’ 5’ Base
-O P O
Base O
-O P O-
O H H
O-
O- H H
H H
H H O H
O H
DNA polymerase O P O-
O P O-
catalyzed O-
Base
Base O
O-
O nucleophilic attack H H
H H
H H
of the 3’-OH on a H
O H
H
O H
phospho-anhydride O P O-
O P O-
Base
O
Base O
O
O H H
H H
H H
H H O H
: OH H
:
O P O-
3’
Base
O
O O O O O O
H H
Base
-O P O P O P O -O P O P OH
O H H
OH H
O- O- O- H H
O- O-
3’
H H
OH H
** Since the 3’ –OH is changed to a –H in ddNTPs, it is unable to form a
phosphodiester bond by nucleophilic attack on the phosphate, and it
will cause a termination in the DNA chain
Sanger Method
• Partial copies of DNA fragments made with DNA
polymerase
• Collection of DNA fragments that terminate with
A,C,G or T using ddNTP
• Separate by gel electrophoresis
• Read DNA sequence
The template DNA pieces are replicated,
incorporating normal nucleotides, but
occasionally and at random dideoxy (DD)
nucleotides are taken up.
This stops replication on that piece of DNA .
The result is a mix of DNA lengths, each ending
with a particular labeled DDnucleotide.
Because the different lengths ‘travel’ at
different rates during electrophoresis, their
order can be determined .
Sanger dideoxy sequencing: basic method
3’ T T T T 5’
5’ 3’
ddA
ddATP in the reaction:
anywhere there’s a T in
ddA the template strand,
ddA
occasionally a ddA will
be added to the
ddA growing strand
Sequencing of DNA by the Sanger method
3’ CCGTAC 5’
primer 5’ 3’
dNTP
ddATP ddTTP ddCTP ddGTP
GGCA GGCAT GGC G
GG
GGCATG
A T C G 3’
the sequence is
complementary to the
original strand
5’
*Originally four separate sets of DNA, primer
and a single different DD nucleotide were
produced and run on a gel.
*Modern technology allows all the DNA,
primers, etc to be mixed and the fluorescent
labeled DDnucleotide ‘ends’ of different
lengths can be ‘read’ by a laser.
*Additionally, the gel slab has been replaced
by polymer filled capillary tubes in modern
equipment .
Gel Separation
• The reaction mixtures are separated on a denaturing
polyacrylamide gel.
– Polyacrylamide can separate the DNA strands which
differ in length by only one nucleotide.
• Each band corresponds to a sequence of DNA which was
terminated by a particular ddNTP.
• This ddNTP is identified by lane in the radioactive method
or by color in the fluorescent method.
• The lowest band on the gel is the shortest. The shorter the
strand, the earlier in the synthetic reaction the ddNTP was
incorporated.
• The lowest band on the gel is at the 5’ end of the new
synthesized strand and is complementary to the 3’ end of
our unknown fragment.
Visualization Methods
• Two forms of labeling:
– Radioactive
• Primer labeled (32P or 33P)
• dNTP labeled (35S or 32P)
– Nonradioactive ( Fluorescence )
*Primer labeled
*ddNTP labeled
- ddNTPs chemically synthesized to contain fluorescence .
- Each ddNTP fluoresces at a different wavelength allowing
identification .
Gel Visualization
• Radioactive method which requires four gel lanes,
one for each reaction vessel.
– Readout is done by hand or with a densitometric
scanner.
• Nonradioactive fluorescence sequencing requires
only one gel lane because each nucleotide has a
distinct color.
– The readout process is done by laser scanner and
recorded by computer.
Gel Electrophoresis and Readout of Reaction Products:
Radioactive vs. Nonradioactive
ddGTP ddATP ddTTP ddCTP
Longest synthesized band =
3’ end of synthesized strand
Sequence of unknown fragment
Sequence of unknown fragment
Shortest synthesized band = 5’ end of synthesized strand
Manual vs. Automatic Sequencing
Comparison list Manual Automatic
DNA labeling Radioisotope Fluorescence dye
Signal detect Film Photomultiplier
Computer
Sample 4 lanes / 1 lane /
1 sequencing sample 1 sequencing sample
Read length 250∼500 bp 400 ∼1000 bp
(average)
Fluorescent Primer Labeled Sequencing
1. Unknown fragment 6. One type of ddNTP per reaction
2. with region of known sequence 7. DNA Polymerase
3. Four separate reactions Why? 8. ddNTP incorporation -
4. Fluorescent labeled primer. stops chain synthesis
9. Run the products on gel. One or four
Different fluorescent dye per
lanes ?
reaction
5. dNTP’s
(dATP, dGTP, dCTP, and dTTP)
ddATP ddGTP ddCTP ddTTP
5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’
Reaction 1 Reaction 2 Reaction 3 Reaction 4
Fluorescent Dideoxynucleotide Labeled Sequencing
1. Here you have one reaction vessel,
with four copies of unknown fragment?
5’ 3’
ddGTP
2. A region of known sequence 3’ 5’
5’ 3’
3. Complementary primer ddATP
4. dNTP’s 3’ 5’
(dATP, dGTP, dCTP, and dTTP) 5’ 3’
ddCTP
5. Fluorescent labeled ddNTP’s. Each
3’ 5’
labeled with a different fluorescent dye
5’ 3’
6. DNA Polymerase ddTTP
7. Again ddNTP incorporation 3’ 5’
stops chain synthesis
One reaction vessel
8. Run the products on gel
Chemical Degradation Method
• Maxam-Gilbert method
– The single stranded DNA fragment to be
sequenced is labeled with radioactive
phosphate at 5’ end .
– The labeled DNA fragment is then divided into four
aliquots, each of which is treated with a reagent
which modifies a specific base .
– This will results in strands of varying lengths.
– Strands separated out with electrophoresis.
– Gels read with radioautography.
Reagents for cleaving DNA
• Aliquot 1 Cleavage at only G
– DNA treated with Dimethyl sulfate (DMS)
– Methylation of G residues at the N7 position
– the glycoside bond of the methylated G residue is hydrolyzed and
the G residue is eliminated.
– Piperidine is added which reacts with hydrolyzed sugar residue,
cleavage of the backbone results
• Aliquot 2 cleavage at G and A
– Use acid instead of DMS
– Position of A revealed
• Aliquot 3: cleavage at C and T
– Treat with hydrazine, then piperidine
• Aliquot 4: cleavage at C only
– Treat with hydrazine in the presence of 1.5 M NaCl
– Position of T revealed
• The four are incubated with piperidine which cleaves the sugar
phosphate backbone of DNA next to the residue that has been modified
Comparison
• Sanger Method • Maxam Gilbert Method
– Enzymatic – Chemical
– Requires little if any – Requires lots of purified
DNA, and many intermediate
DNA purification purification steps
– No labeling of the DNA – labeling of the DNA strand
sequencing template – Requires long stretches of
– Requires DNA synthesis DNA
– Breaks DNA at different
– Termination of chain nucleotides
elongation – Relatively short readings
– Automation is available – Automation not available
(sequencers) (sequencers)