DNA SEQUENCING

TO-; By;-
Dr. Manjeet Kaur KANIKA
1603
Biotech. 5th semester
U.I.E.T. M.D.U.
 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
harmful effect in a plant or animal.
 DNA Sequence variation can change the Protein produced by a
particular gene.
Gene A Codon resul t s in GCA AGA GAT AAT TGT
from part icu lar Am in o Ala Arg Asp Asn Cys
per son 1 Acid (AA) sequ ence

Gene A Codon chan ge GCG AGA GAT AAT TGT

from m akes no differen ce
per son 2 in AA Ala Arg Asp Asn Cys
( redu n dan t code)
Gene A Codon chan ge GCA AAA GAT AAT TGT
from resul t s in diff erent Ala Lys Asp Asn Cys
per son 3 AA sequ en ce

•Simple point mutations such as this can cause altered protein shape
and function.
•Diseases such as Sickle Cell Anaemia and Cystic Fibrosis are caused by
point mutations
•Historically there are two main methods of DNA
sequencing:

•Maxam & Gilbert,using chemical sequencing

•Sanger, using dideoxynucleotides.

Modern sequencing equipment uses the principle

of the Sanger technique.
 Methods for DNA sequencing

• Sanger’s or Chain Termination Method.

• Automated DNA Sequencing.
• Maxam and Gilbert Chemical degradation method.
• Using Bacteriophage M13 as Vector.
• Primer walking.
• Direct DNA Sequencing using PCR.
 MAXAM & GILBERT DNA SEQUENCING
(CHEMICAL DEGRADATION)
In the late 1970s, A. M. Maxam and W.Gilbert devised the
first method for sequencing DNA fragments containing up
to ≈500 nucleotides.

 The sequence of a double-stranded DNA molecule isdetermined

by treatment with chemicals that cut the molecule at specific
nucleotide position.

 It is the early method involving base-specific chemical

modification and subsequent cleavage of DNA.

 Most of the chemicals used in chemical degradation method are

toxic and hazardous to the health of the researchers doing the
DNA sequencing.
 The Maxam-Gilbert Method
• Includes chemical degradation of DNA.
• If template DNA is able to form intrastrand base
pair then it might be impossible to obtain
accurate sequence by Chain Termination
Method..
• Intrastrand base pairs block activity of DNA
polymerase.
• This alter mobility of chain terminated molecules
during electrophoresis.
 Requirements(Maxam gilbert method)
• Double stranded DNA.
Requirements(Maxam gilbert method)

• No need of Primers.

• Cleavage reagents
Most of reagents used are very toxic chemicals
that cleave DNA molecule .
 CHEMICALS INVOLVED
 Dimethyl sulphate methylates guanine.

Acid removes any purines.

Hydrazine modifies any pyrimidine.

 Hydrazine with NACL specifically modifies cytosines.

 Piperidine is used to remove the modified bases.

 PROCEDURE
Double stranded DNA fragment is labelled by attaching radioactive phosphorus group to
5’ end

Dimethyle sulphoxide is added and labelled DNA sample heated to 90̊⁰c.

(Base pairing breaks and DNA
become single stranded)

Four samples are taken and treated with cleavage reagents.

Electrophoresis
 CHEMICAL DEGRADATION METHOD
PROCEDURE

Four samples of an end-labeled DNA restriction fragment are chemically

cleaved at different specific nucleotides.

The resulting sub-fragments are separated by agarose gel

electrophoresis and the labeled fragments are detected by
autoradiograph.

the sequence of the original end-labeled restriction fragment

can be determined directly from parallel electrophoretogram
of the four samples.
• First set of reagents added cause chemical modification
in nucleotides for which they are specific making
strand susceptible to cleavage at that nucleotide when
an additional chemical PIPERIDINE is added.

• Some of cleaved fragment retain P32 label at their 5’

end. After electrophoresis ,the bands visualised by
autoradiograpy represent these labelled fragments.

• The nucleotide sequence can be read from

autoradiography.
 Frederick Sanger
Discovered DNA sequencing by chain termination
method
• Nobel Prize 1 (1958)
– Complete amino acid
sequence of insulin
• Nobel Prize 2 (1980)
– For DNA sequencing
 SANGER METHOD
 F. Sanger and his colleagues developed a second
method of DNA sequencing, which now is used
much more frequently than the Maxam-Gilbert method.

 The sequence of a single-stranded DNA molecule is

determined by enzymatic synthesis of complementary
polynucleotide chains, these chains terminating specific

Nucleotide position.

 Sanger method is the most suitable method for

automation in large scale sequencing projects, and most
general sequencing is now carried out in this way.
 The Sanger method requires
• Multiple copies of single stranded template DNA
• A suitable primer (a small piece of DNA that can
pair with the template DNA to act as a starting
point for replication)
• DNA polymerase (an enzyme that copies DNA,
adding new nucleotides to the 3’ end of the
template
• A ‘pool’ of normal nucleotides
• A small proportion of dideoxynucleotides labeled in
some way ( radioactively or with fluorescent dyes)
 The Sanger Technique
• Uses dideoxynucleotides
(dideoxyadenine, dideoxyguanine, etc)
• These are molecules that resemble
normal nucleotides but lack the normal
-OH group.
 Dideoxy Nucleotides

• Lack an -OH group at

the 3-carbon position
• Cannot add another
nucleoside at that
position
• Prevent further DNA
synthesis
• Because they lack the -OH (which allows
nucleotides to join a growing DNA strand),
replication stops.
Normally, this would
be where another phosphate
Is attached, but with no -OH
group, a bond can not form
and replication stops
WHY SANGER METHOD TERMED AS
CHAIN TERMINATION METHOD????

Specific terminators of DNA Chain Elongation is 2’3’-dideoxy

nucleoside triphosphates (ddNTPs) can be incorporated

normally into a growing DNA chain through their
5’triphosphate groups.

 ddNTPs are telogens ,they can not form phospho -diester bonds

with the next incoming deoxynucleotide (dNTPs).

 Nucleotides which causes chain termination because they

lack 3’ hydroxyl group for extension .hence this technique
called as dideoxy (or) chain termination method.
 CHAIN TERMINATION METHOD
PRINCIPLE
The single-stranded DNA to be sequenced serves as the
template strand for in vitro DNA synthesis; a synthetic
5′-end-labeled oligodeoxynucleotide is used as the primer.

 When a small amount of a specific dideoxy NTPs(ddNTPs)

is included along with the four deoxy NTPs normally
required in the reaction mixture for DNA polymerase.

 The products are the series of chains that are specifically

terminated at the dideoxy residue.

 Thus four separate reactions, each containing a different

dideoxy NTP,can be run, and their products displayed on
high-resolution Acryl amide gel.
 CHAIN TERMINATION METHOD

PROCEDURE
Prepared the starting material for a chain termination sequencing
experiment is a identical single-stranded DNA molecules..

To anneal a short oligonucleotide to the same position on each molecule, this

oligonucleotide subsequently acting as the primer for synthesis of a new DNA
strand that is complementary to the template catalyzed by DNA polymerase
requires requires the four deoxyribonucleotide triphosphates (dNTPs - dATP,
dCTP, dGTP and dTTP).

The polymerase does not discriminate between dNTPs and ddNTPs, so the
dideoxynucleotide can be incorporated into the growing chain, but it then
blocks further elongation because it lacks the 3′-hydroxyl group needed to
form a connection with the nucleotide.
This process continue until several hundred nucleotides have been
polymerized before a ddATP is eventually incorporated. The result
is therefore a set of new chains, all of different lengths, but each
ending in ddATP.

Now the polyacrylamide gel comes on to play. The family of the

presence of ddNTPAs (ddATP,ddCTP,ddGTP,ddTTP) loaded into
four adjacent wells ofthe gel.

After electrophoresis, the DNA sequence can be read directly from

the positions of the bands in the gel.
•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.
• 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.
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
Sanger sequencing
• DNA is fragmented
• Cloned to a plasmid
vector
• Cyclic sequencing
reaction
• Separation by
electrophoresis
• Readout with
fluorescent tags
CHAIN TERMAINTAION METHOD
THE DNA CAN BE CLONED IN A BACTERIOPHAGE M13
VECTOR

M13 bacteriophage has a single-stranded DNA genome which,

after infection of Escherichia coli bacteria, is converted into a
double-stranded replicative form.

 At the same time the infected cells continually secrete new M13
phage particles, approximately 1000 per generation ,these
phages containing the single-stranded version of the genome.

 The one disadvantage is that DNA fragments longer than about

3 kb suffer deletions and rearrangements when cloned in an
M13 vector, so the system can only be used with short pieces of

DNA.
 PROCEDURE
1. M13 infective particles are released from host then
from M13 genome non essential region is removed
and cloned DNA is inserted.
2. Recombinant DNA is obtained which is taken by
competent E.Coli cells.
3. After incubation plating and is done and cells are
grown on media containing X gal.
4. Blue and white colonies are obtained.
white colonies contain target DNA.
5. DNA is isolated from colonies and used as source of
single stranded DNA.
6. To S.S.DNA primers and polymerase is added
7. Visualised by autoradiography.
PCR CAN BE USED TO GENERATE SINGLE
STRANDED DNA .
•One way of using PCR to prepare
template DNA for chain
termination sequencing.

•The PCR is carried out with one

normal primer (shown in red), and
one primer that is labeled with a
metallic bead (shown in brown).

•After PCR, the labeled strands are

purified with a magnetic device.
 The discovery of thermo stable DNA polymerases, which led to
the development of PCR has also resulted in new methodologies
for chain termination sequencing.

 Thermal cycle sequencing has two advantages over traditional

chain termination sequencing –
1) uses double-stranded rather than single-stranded DNA as the
starting material.
2) very little template DNA is needed, so the DNA does not have to
be cloned before being sequenced.

one primer is used and each reaction mixture includes

one of the ddNTP. Because there is only one primer, only one of
the strands of the starting molecule is copied, and the product
accumulates in a linear fashion
 The presence of the ddNTP in the reaction mixture causes chain
termination strands can be analyzed and the sequence read in the
normal manner by polyacrylamide gel electrophoresis.
Thermal cycle sequencing.

PCR is carried out with just one

primer and with a
dideoxynucleotide present in the
reaction mixture.

The result is a family of chain-

terminated strands - the ‘A'
family in the reaction shown.

These strands, along with the

products of the C,GandT
reactions, are electrophoresed as
in the standard methodology.
 AUTOMATED DNA SEQUENCING
(Leroy Hood and colleagues,1986)

The most dramatic advance in sequencing was the introduction

of automated sequencing using fluorescence-labeled dideoxy-

terminators.

 In their method, the primer was labeled with one of four

different fluorescent dyes and each was placed in a separate
sequencing reaction with one of the four dideoxynucleotides
plus all four deoxynucleotides.

 Fluorolabeling has been equally important in the development

of sequencing methodology, in particular because the
detection system for fluorolabels has opened the way to
automated sequence reading.
 AUTOMATED DNA SEQUENCING.
• ADS is carried out as chain termination method but
Fluorescent labels are used instead of radioactive
labelling.
• Fluorescent labells are attached to dideoxynucleotides
so chain terminated molecules carries single labell at its
3’ end.
• Different fluorochrome can be used for each of four
nucleotide.
• The fluorecent detector identifies signal emitted by
each band and feeds the data to computer which
convert information into appropriate DNA sequence.
 DNA sequencing
– Automated DNA sequencing – in automated DNA
sequencing a radioactive deoxynucleotide is not used
and all four dideoxy reactions are done in a single tube.
– This is possible because each ddNTPs is labeled with a
different flourescent dye.
– Therefore the dye present in each synthesized fragment
corresponds to the dye attached to the
dideoxynucleotide that was added to terminate the
synthesis of that particular fragment.
– The contents of the single tube reaction are loaded
onto a single lane of a gel and electrophoresis is done.
ADVANTAGES of Automated DNA sequencing
• Autoradiograph sequence is difficult to read as
compared to fluorescent detection.
• In single lane all four nucleotides can be added.
• Good quality data is obtained.
• Output is in machine readable form.
• Large amount of data can be sequenced upto
750 nucleotides.
• Errors are eliminated.
AUTOMATED DNA SEQUENCING WITH FLUORESCENTLY
LABELED DIDEOXYNUCLEOTIDES
 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.
Fluorescent end labeling of DNA
 Sequencing by PRIMER WALKING.

• When sequencing large amount of DNA

primer walking is used.

• Cloning is done in bacteriophage lambda or in

cosmid vector.
 Target DNA
(Linked with plasmid DNA)

Purified and anealed with oligonucleotide primer.

(anealing occur by base pairing)

Dideoxy nucleotide sequence reaction.

Identify order of first 250-300 nucleotides.

(insert second primer)

Second primer hybridize 300 ntd. Away from 1st primer.

 repeat reaction.

detect sequence of next 300 ntd.

(3rd primer is added)

Repeat reaction

Full DNA sequence is determined.

ADVANTAGES;
•Eliminates error
•Exact anealing with DNA occur.
 A sequencing gel

This picture is a radiograph. The dark color of the lines is

proportional to the radioactivity from 32P labeled adenonsine
in the transcribed DNA sample.
THANKS
Wilson.G.Anandaraj
I M.Sc Environmental Biotechnology
Bharadhidasan University.
FOR CONTACT: anandaraj.wilson@gmail.com
 WHAT IS DNA SEQUENCING ?

 DNA sequencing usually involves enzymatic DNA

synthesis in the presence of base-specific
dideoxynucleotide chain terminators.

 Determining the DNA sequence is therefore useful in

basic research studying fundamental biological
processes, as well as in applied fields such as
diagnostic or forensic research.
 FOUNDERS OF SEQUENCING TECHNOLOGY

Sanger Wally Gilbert

 MAXAM & GILBERT DNA SEQUENCING
(CHEMICAL DEGRADATION)
 In the late 1970s, A. M. Maxam and W.Gilbert
devised the first method for sequencing DNA
fragments containing up to ≈500 nucleotides.

 the sequence of a double-stranded DNA molecule is

determined by treatment with chemicals that cut the
molecule at specific nucleotide positions.

 it is the early method involving base-specific chemical

modification and subsequent cleavage of DNA.

 most of the chemicals used in chemical degradation

method are toxic and hazardous to the health of the
researchers doing the DNA sequencing.
CHEMICAL DEGRADATION METHOD

PROCEDURE

four samples of an end-labeled DNA restriction fragment are

chemically cleaved at different specific nucleotides.

the resulting sub-fragments are separated by agarose

gel electrophoresis and the labeled fragments are
detected by autoradiograph.

the sequence of the original end-labeled restriction fragment

can be determined directly from parallel electrophoretograms
of the four samples.
 CHEMICALS INVOLVED

 Dimethyl sulphate methylates guanine.

 Acid removes any purines.

 Hydrazine modifies any pyrimidine.

 Hydrazine with NACL specifically modifies

cytosines.

 Piperidine is used to remove the modified bases.

 SANGER METHOD

 F. Sanger and his colleagues developed a second

method of DNA sequencing, which now is used
much more frequently than the Maxam-Gilbert method.

 The sequence of a single-stranded DNA molecule is

determined by enzymatic synthesis of complementary
polynucleotide chains, these chains terminating at
specific
nucleotide positions.

 Sanger method is the most suitable method for

automation in large scale sequencing projects, and most
general sequencing is now carried out in this way.
WHY SANGER METHOD TERMED AS
CHAIN TERMINATION METHOD????

 Specific terminators of DNA Chain Elongation is

2’3’-dideoxy nucleoside triphosphates (ddNTPs)
can be incorporated normally into a growing DNA
chain through their 5’triphosphate groups.

 ddNTPs are telogens ,they can not form phospho -

diester bonds with the next incoming deoxynucleotides
(dNTPs).

 Nucleotides which causes chain termination because

they
lack 3’ hydroxyl group for extension .hence this
technique
called as dideoxy (or) chain termination method.
 CHAIN TERMINATION METHOD

PRINCIPLE

 The single-stranded DNA to be sequenced serves as the

template strand for in vitro DNA synthesis; a synthetic
5′-end-labeled oligodeoxynucleotide is used as the primer.

 When a small amount of a specific dideoxy NTPs (ddNTPs)

is included along with the four deoxy NTPs normally
required in the reaction mixture for DNA polymerase.

 The products are the series of chains that are specifically

terminated at the dideoxy residue.

 Thus four separate reactions, each containing a different

dideoxy NTP,can be run, and their products displayed on a
high-resolution Acryl amide gel.
 CHAIN TERMINATION METHOD

PROCEDURE

Prepared the starting material for a chain termination sequencing experiment is a

identical single-stranded DNA molecules.

To anneal a short oligonucleotide to the same position on each molecule, this

oligonucleotide subsequently acting as the primer for synthesis of a new DNA
strand that is complementary to the template catalyzed by DNA polymerase
requires requires the four deoxyribonucleotide triphosphates (dNTPs - dATP,
dCTP, dGTP and dTTP) as substrates, would normally continue until several
thousand nuceotides had been polymerised.

The polymerase does not discriminate between dNTPs and ddNTPs, so the
dideoxynucleotide can be incorporated into the growing chain, but it then block
further elongation because it lacks the 3′-hydroxyl group needed to form a
connection with the nucleotide.
 (Continue)

This process continue until several hundred nucleotides have been polymerized
before a ddATP is eventually incorporated. The result is therefore a set of new
chains, all of different lengths, but each ending in ddATP.

Now the polyacrylamide gel comes on to play. The family of the presence of
ddNTPAs(ddATP,ddCTP,ddGTP,ddTTP) loaded into four adjacent wells of
the gel.

After electrophoresis, the DNA sequence can be read directly from the positions
of the bands in the gel.
CHAIN TERMAINTAION METHOD
 HOW DOES THE DNA TEMPLATE OBTAINED?

The template for a chain termination experiment is a

single-stranded version of the DNA molecule to be
sequenced. There are several ways in which this can be
Obtained;

 The DNA can be cloned in a plasmid vector.

 The DNA can be cloned in a bacteriophage M13 vector.

 The DNA can be cloned in a phagemid.

 PCR can be used to generate single-stranded DNA.

 THE DNA CAN BE CLONED IN A PLASMID VECTOR

 The double stranded plasmid DNA strand must be converted

into single-stranded DNA by denaturation with alkali or by
boiling. This is a common method for obtaining template
DNA for DNA sequencing largely.

 A single strand of plasmid is that it can be difficult to prepare

plasmid DNA that is not contaminated with small quantities
of bacterial DNA and RNA, which can act as spurious templates
or primers in the DNA sequencing experiment.
THE DNA CAN BE CLONED IN A BACTERIOPHAGE M13
VECTOR

 M13 bacteriophage has a single-stranded

DNA genome which,after infection of
Escherichia coli bacteria, is converted into a
double-stranded replicative form.

 At the same time the infected cells

continually secrete new M13 phage
particles, approximately 1000 per
generation ,these phages containing the
single-stranded version of the genome.

 The one disadvantage is that DNA

fragments longer than about 3 kb suffer
deletions and rearrangements when
cloned in an M13 vector, so the system
can only be used with short pieces of DNA.
PCR CAN BE USED TO GENERATE
SINGLE STRANDED DNA

One way of using PCR to prepare template DNA for chain

termination sequencing. The PCR is carried out with one
normal primer (shown in red), and one primer that is
labeled with a metallic bead (shown in brown). After PCR
the labeled strands are purified with a magnetic device.
 RECENT INNOVATIONS IN CHAIN
TERMINATION SEQUENCING

Thermal cycle sequencing

 Automated DNA sequencing

 Pyrosequencing

 Sequencing by hybridization
 THERMAL CYCLE SEQUENCING
( Sears et al., 1992)
 The discovery of thermo stable DNA polymerases, which led to the
development of PCR has also resulted in new methodologies for chain
termination sequencing.

 Thermal cycle sequencing has two advantages over traditional chain

termination sequencing
1) uses double-stranded rather than single-stranded
DNA as the starting material.
2) very little template DNA is needed, so the DNA does
not have to be cloned before being sequenced.

 Thermal cycle sequencing is carried out in a similar way to PCR but

just
one primer is used and each reaction mixture includes one of the
ddNTP. Because there is only one primer, only one of the strands of the
starting molecule is copied, and the product accumulates in a linear
fashion, not exponentially as is the case in a real PCR.

 The presence of the ddNTP in the reaction mixture causes chain

termination, as in the standard methodology, and the family of
THERMAL CYCLE SEQUENCING

Thermal cycle sequencing.

PCR is carried out with just one primer and with a

dideoxynucleotide present in the reaction mixture. The resul
is a family of chain-terminated strands - the ‘A' family in the
reaction shown. These strands, along with the products of th
C, G and T reactions, are electrophoresed as in the standard
methodology
 AUTOMATED DNA SEQUENCING
(Leroy Hood and colleagues,1986)
 The most dramatic advance in sequencing and the one that
carried DNA sequencing into a high throughput environment
was the introduction of automated sequencing using
fluorescence-labeled dideoxy-terminators.

 In 1986, Leroy Hood and colleagues reported on a DNA

sequencing method in which the radioactive labels,
autoradiography, and manual base calling were all replaced
by fluorescent labels, laser induced fluorescence detection,
and computerized base calling.

 In their method, the primer was labeled with one of four different
fluorescent dyes and each was placed in a separate sequencing
reaction with one of the four dideoxynucleotides plus all four
deoxynucleotides.

 Fluorolabeling has been equally important in the development of

sequencing methodology, in particular because the detection system
for fluorolabels has opened the way to automated sequence reading.
AUTOMATED DNA SEQUENCING WITH FLUORESCENTLY
LABELED DIDEOXYNUCLEOTIDES

(A) The chain termination reactions are carried out in a

single tube, with each dideoxynucleotide labeled
with a different fluorophore.In the automated
sequencer, the bands in the electrophoresis gel
move past a fluorescence detector, which identifies
which dideoxynucleotide is present in each band.
The information is passed to the imaging system.

(B) The printout from an automated sequencer.

The sequence is represented by a series of peaks,
one for each nucleotide position. In this example,
a green peak is an ‘A', blue is ‘C', black is ‘G',
and red is ‘T'.
 PYROSEQUENCING

 A novel DNA sequencing method in which addition of a nucleotide to the

end of a growing polynucleotide is detected directly by conversion of the
released pyrophosphate into a flash of chemiluminescence's.

 Does not require electrophoresis or any other fragment separation

procedure and so is more rapid than chain termination sequencing.

Template is copied in a straightforward manner without added ddNTPs.

 During sequencing the addition of a nucleotide to the end of the growing

strand is detectable because it is accompanied by release of a molecule
of pyrophosphate, which can be converted by the enzyme sulfurylase
into a flash of chemiluminescence's.

 Each dNTP is therefore added separately, one after the other, with a
nucleotidase enzyme also present in the reaction mixture so that if a
dNTP is not incorporated into the polynucleotide then it is rapidly
degraded before the next dNTP is added.
PYROSEQUENCING

The strand synthesis reaction is carried out in the

absence of dideoxynucleotides. Each dNTP is added
individually, along with a nucleotidase enzyme that
degrades the dNTP if it is not incorporated into the
strand being synthesized. Incorporation of a nucleotide
is detected by a flash of chemiluminescence induced
by the pyrophosphate released from the dNTP. The
order in which nucleotides are added to the growing
strand can therefore be followed.
 SEQUENCED BY HYBRIDIZATION

The Hybridization technique is a very different approach to DNA

sequencing through the use of DNA chips might one day be possible.

 A chip carrying an array of different oligonucleotides could be used in

DNA sequencing by applying the test molecule.

 Hybridization to an individual oligonucleotide would indicate the

presence of that particular oligonucleotide sequence in the test
molecule, and comparison of all the oligonucleotides to which
hybridization occurs would enable the sequence of the test molecule
to be deduced.

 The problem with this approach is that the maximum length of the
molecule that can be sequenced is given by the square root of the
number of oligonucleotides in the array.
SEQUENCED DY HYBRIDIZATION

The chip carries an array of every possible

8-mer oligonucleotide.

 The DNA to be sequenced is labeled with a

fluorescent marker and applied to the chip, and the
positions of hybridizing oligonucleotides determined
by confocal microscopy.

 Each hybridizing oligonucleotide represents an

8-nucleotide sequence motif that is present in the
probe DNA.

 The sequence of the probe DNA can therefore be

deduced from the overlaps between the sequences
of these hybridizing oligonucleotides.
RECENT TRENDS IN DNA SEQUENCING
BIOINFORMATICS TOOLS FOR DNA SEQUENCING