Nobel Laureates

Walter Gilbert
Paul Berg
Sanger
Institute
1953 Discovery of the structure of the DNA double helix.
1972 Development of recombinant DNA technology, which permits
isolation of defined fragments of DNA; prior to this, the only accessible
samples for sequencing were from bacteriophage or virus DNA.
1975 The first complete DNA genome to be sequenced is that of
bacteriophage X174
1977 Allan Maxam and Walter Gilbert publish "DNA sequencing by
chemical degradation". Frederick Sanger, independently, publishes "DNA
sequencing by enzymatic synthesis".
1980 Frederick Sanger and Walter Gilbert receive the Nobel Prize in Chemistry
Historical facts in DNA sequencing
Historical facts in DNA sequencing
1984 Medical Research Council scientists decipher the complete DNA
sequence of the Epstein-Barr virus, 170 kb.
1986 Leroy E. Hood's laboratory at the California Institute of Technology
and Smith announce the first semi-automated DNA sequencing machine.
1987 Applied Biosystems markets first automated sequencing machine, the
model ABI 370.
1990 The U.S. National Institutes of Health (NIH) begins large-scale
sequencing trials on Mycoplasma capricolum, Escherichia coli,
Caenorhabditis elegans, and Saccharomyces cerevisiae (at 75 cents
(US)/base).
Basic research studying
fundamental biological
processes,
In applied fields
diagnostic
forensic research
MAXAM & GILBERT
DNA SEQUENCING
I n t he l at e 1970s, A. M. Maxam and W.
Gi l ber t devi sed t he f i r st met hod f or
sequenci ng DNA f r agment s cont ai ni ng up t o
500 nucl eot i des.

Ear l y met hod i nvol vi ng base- speci f i c
chemi cal modi f i cat i on and subsequent
cl eavage of DNA.

Toxi c and hazar dous chemi cal s used.

CHEMICALS INVOLVED

Dimethyl sulphate methylates
guanine.
Acid removal of purines.
Hydrazine modifies any pyrimidine.
Hydrazine with NaCl (1.5M)
specifically modifies cytosines.
Piperidine is used to remove the
modified bases.

The sequence of the original end-labeled restriction
fragment can be determined directly from parallel
electrophoretograms of the four samples.
The resulting sub-fragments are separated by agarose gel
electrophoresis and the labeled fragments are detected by
autoradiograph.
Four samples of an end-labeled DNA restriction fragment
are chemically cleaved at different specific nucleotides.
CHEMICAL DEGRADATION METHOD

Cl eavage at G usi ng di met hyl sul f at e,
f ol l owed by st r and sci ssi on wi t h pi per i di ne

Dimethyl sulfate reacts with guanine to methylate it at
the 7-position.
Leads to instability of the N-9 glycosidic bond
(Under alkaline conditions)
Purine ring is degraded and released. (In the
presence of OH
-
and the secondary
amine piperidine)


A -elimination reaction facilitated by
piperidine
Causes the excision of the naked
deoxyribose moiety from the sugarphosphate
backbone
Consequent scission of the DNA strand to
yield 5'- and 3'- fragments.
Cleavage at A or G:
DNA i s f i rst t reat ed wi t h aci d
Di met hyl sul f at e met hyl at es
adeni ne at t he 3- posi t i on as wel l as
guani ne at t he 7- posi t i on
Subsequent react i on
Tri ggers degradat i on and
di spl acement of t he met hyl at ed A
or G puri ne base and st rand
sci ssi on
Hydrolysis of pyrimidine rings by
hydrazine
Hydr azi ne ( H
2
N- NH
2
) at t acks acr oss t he C-
4 and C- 6 at oms of pyr i mi di nes t o open
t he r i ng
Subsequent modi f i c at i on of t he deox y r i bose,
r ender i ng i t s us c ept i bl e t o - el i mi nat i on
5' - and 3' - f r agment s ar e pr oduced.
The pr esence of hi gh sal t concent r at i ons
pr ot ect s T ( but not C) f r om r eact i on wi t h
hydr azi ne.

A band in the lanes corresponding to the
C only and the C + T reactions would be
called a C.
If the band was present in the C + T
reaction lane but not in the C only
reaction lane it would be called a T.
The same decision process would obtain
for the G only and the G + A reaction
lanes.
- interpreting the banding pattern relative to
the four chemical reactions.
Limitations
Technical complexity
prohibiting its use in
standard molecular
biology kits
Extensive use of
hazardous chemicals
Difficulties with
scale-up
ddNTPs are telogens
They can not form phospho -
diester bonds with the next
incoming deoxynucleotides
(dNTPs).
Lack 3 hydroxyl group for
extension
2 3 - di de o x y nuc l e o s i de
t r i pho s pha t e s ( ddNTPs )
S pe c i f i c t e r mi na t o r s o f DNA
Cha i n El o ng a t i o n
Ca n b e i nc o r po r a t e d no r ma l l y
i nt o a g r o wi ng DNA c h a i n
t h r o ug h t h e i r 5 t r i pho s pha t e
g r o ups .
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.
Also known as the primed synthesis method

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.

DNA polymerase used:
Usually Sequenase 2

, a genetically
engineered version of bacteriophage T7
DNA polymerase that lacks all traces of
exonuclease activity that might
otherwise degrade the DNA

Starting material:
Identical single-stranded DNA
molecules of unknown sequence
CHAIN TERMINATION
METHOD
Major Steps

1. Template DNA (ssDNA)
2. Primer annealing
3. Complementary strand synthesis
4. Labeling for the detection of fragments
5. Chain termination using ddNTPs
6. Resolution on denaturing PAGE
7. Visualization of bands by autoradiography

Steps
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 complementary to the template

Catalyzed by DNA polymerase using the four
deoxyribonucleoside triphosphates (dNTPs) and the
corresponding ddNTPs
The polymerase does not discriminate between dNTPs and ddNTPs

Dideoxynucleotide can be incorporated into the growing chain

It then blocks further elongation because it lacks the 3-
hydroxyl group needed to form a connection with the
nucleotide.
Now the polyacrylamide gel comes on to play. The reaction
mixtures each containing one of the ddNTPs
(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.

The 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.

Advantages over chemical cleavage method
Chain termination-based
kits are commercially
available that contain the
reagents needed for
sequencing, pre-aliquoted
and ready to use.
Chain-
termination
methods have
greatly simplified
DNA sequencing.
Obtaining the dna templ ate
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.

Starting material can be any circular template from a
colony, culture, glycerol stock, or plaque.

A drawback of using plasmid is that it may 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 is shown

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.

Cycle sequencing is a modification of the traditional
Sanger sequencing method.
The principles are the same as in Sanger
sequencing; Dideoxynucleotides are used in a
polymerization reaction to create a nested set of DNA
fragments with dideoxynucleotides at the 3' terminus
of each fragment.
The key difference is that cycle sequencing employs
a thermostable DNA polymerase which can be heated
to 95
o
C and still retain activity.
Cycle sequencing
Cycle sequencing
The advantage of using such a polymerase is that the
sequencing reaction can be repeated over and over
again in the same tube by heating the mixture to
denature the DNA and then allowing it to cool to anneal
the primers and polymerize new strands.

Thus, less template DNA is needed than for
conventional sequencing reactions.
Furthermore, the repeated heating and cooling can be
done in a DNA thermal cycler.

Cycle sequencing

Advantages:
Works with ssDNA and dsDNA and thus eliminates the
need for M13 phage
Requires only small amounts of template
Can be set up in microtitre plates or microtubes
Can use internal labeling with [-32P], [-33P], or [35S]or
with 5- end labeled primer
Can be adapted for rapid screening

Thermal cycle
sequencing
Automated
DNA
sequencing
Pyrosequencing
Sequencing by
hybridization





RECENT
INNOVATIONS
IN CHAIN
TERMINATION
SEQUENCING







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 ( Sears et al., 1992)


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,
The family of resulting strands can be analyzed and the sequence
read in the normal manner by polyacrylamide gel electrophoresis











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.






AUTOMATED DNA SEQUENCING
(Leroy Hood and
colleagues,1986)
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'.





A n o v e l DNA s e q u e n c i n g me t h o d i n w h i c h a d d i t i o n o f a n u c l e o t i d e
t o t h e e n d o f a g r o w i n g p o l y n u c l e o t i d e i s d e t e c t e d d i r e c t l y b y
c o n v e r s i o n o f t h e r e l e a s e d p y r o p h o s p h a t e i n t o a f l a s h o f
c h e mi l u mi n e s c e n c e ' s .
Do e s n o t r e q u i r e e l e c t r o p h o r e s i s o r a n y o t h e r f r a g me n t s e p a r a t i o n
p r o c e d u r e
S o i s mo r e r a p i d t h a n c h a i n t e r mi n a t i o n s e q u e n c i n g .
T e mp l a t e i s c o p i e d i n a s t r a i g h t f o r w a r d ma n n e r w i t h o u t a d d e d
d d NT Ps .
Du r i n g s e q u e n c i n g t h e a d d i t i o n o f a n u c l e o t i d e t o t h e e n d o f t h e
g r o w i n g s t r a n d i s d e t e c t a b l e b e c a u s e i t i s a c c o mp a n i e d b y r e l e a s e o f a
mo l e c u l e o f p y r o p h o s p h a t e , w h i c h c a n b e c o n v e r t e d b y t h e e n z y me
s u l f u r y l a s e i n t o a f l a s h o f c h e mi l u mi n e s c e n c e ' s .
Ea c h d NT P i s t h e r e f o r e a d d e d s e p a r a t e l y , o n e a f t e r t h e o t h e r , w i t h a
n u c l e o t i d a s e e n z y me a l s o p r e s e n t i n t h e r e a c t i o n mi x t u r e s o t h a t i f a
d NT P i s n o t i n c o r p o r a t e d i n t o t h e p o l y n u c l e o t i d e t h e n i t i s r a p i d l y
d e g r a d e d b e f o r e t h e n e x t d NT P i s a d d e d .





PYROSEQUENCING





SEQUENCING 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.





SEQUENCING BY
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
Be g u n f o r ma l l y i n 1 9 9 0
Co o r di n a t e d by t h e U. S . De pa r t me n t o f
E n e r g y a n d t h e Na t i o n a l I n s t i t u t e s o f
He a l t h .
Wa s pl a n n e d t o l a s t 1 5 y e a r s , bu t r a pi d
t e c h n o l o g i c a l a dv a n c e s a c c e l e r a t e d t h e
c o mpl e t i o n da t e t o 2 003 .
I de n t i f y a l l t h e a ppr o x i ma t e l y 2 0, 000-
2 5 , 000 ge n e s i n h u ma n DNA,
De t e r mi n e t h e s e q u e n c e s o f t h e 3 bi l l i o n
c h e mi c a l ba s e pa i r s t h a t ma ke u p h u ma n
DNA,
S t o r e t h i s i n f o r ma t i o n i n da t a ba s e s ,
I mpr o v e t o o l s f o r da t a a n a l y s i s ,
T r a n s f e r r e l a t e d t e c h n o l o g i e s t o t h e
pr i v a t e s e c t o r , a n d
Addr e s s t h e e t h i c a l , l e g a l , a n d s o c i a l i s s u e s
( E L S I ) t h a t ma y a r i s e f r o m t h e pr o j e c t .
CLONE
Hap Map
Project
T h e I n t e r na t i ona l Ha pMa p P r oj e c t i s a n o r ga ni z a t i on
whos e go a l i s t o de v e l op a h a pl ot y pe ma p o f t h e h u ma n
ge n ome ( t h e Ha pMa p) , whi c h wi l l de s c r i be t h e c o mmon
pa t t er n s o f h u ma n ge n et i c v a r i a t i on.

T h e Ha pMa p i s e x pe c t e d t o be a k e y r e s our c e f or
r e s e a r c he r s t o f i n d ge n e t i c v a r i a n t s a f f e c t i ng h e a l t h,
di s e a s e a n d r e s pon s e s t o dr ugs a n d e n v i r on me nt a l
f a c t or s .

T h e i n f or ma t i on pr oduc e d by t h e pr oj e c t i s ma de f r e e l y
a v a i l a bl e t o r e s e a r c he r s a r oun d t h e wor l d
Date
Cost per Mb of DNA
Sequence
Cost per Genome
September-
2001
$ 5,292.39 $ 95,263,072
October-2010 $ 0.32 $ 29,092
Futuristic methods of DNA Sequencing
Most next-
generation DNA
sequencing
methods have
focused on
either :
Template
amplification
followed by
massively-parallel
sequencing-by-
synthesis
Single molecule
detection.
The first method is
now commercially
available but suffers
from relatively large
volumes of expensive
reagent usage.
The latter method will
have a disadvantage in
signal-to-noise and
therefore require more
sensitive and expensive
instrumentation.
Microfluidic DNA
Sequencing
A single molecule
detection method
leveraging droplet-based
microfluidics.
This should limit the
amount of reagent
required to sequence
DNA to less than several
milliliters, while still
retaining the ability to
amplify the template that
thereby enables us to use
relatively inexpensive and
robust detection.
The
method
is simple
and
does not
require
enzymes
.
Futuristic methods of DNA Sequencing
Polony Sequencing
Ultra-high throughput polony genome sequencing, generating
raw data to re-sequence the human genome in one week.
The goal of the method is to increase the polony sequencing read length
using a cyclic ligation strategy that involves enzymatic cleavage, and
increase read density by using different clonal amplification strategies.
Futuristic methods of DNA Sequencing
Step 1:
Make library of linear DNA molecules
with universal priming sites
Step 2:
Amplify polymerase colonies
(polonies) in a polyacrylamide gel
Step 3:
Sequence polonies by fluorescent in
situ sequential quantitation (FISSEQ).
Millikan Sequencing by Label-Free Detection of
Nucleotide Incorporation


This measures the increased charge as nucleotides are added to DNA
templates attached to a tethered bead.

Opposing electrical, hydrodynamic and entropic forces will be used to
measure the bead displacement, which is a function of the length of DNA
attached to the bead.

Much lower per-bead copy number required

enable amplification options other than emulsion PCR, such as bridge
PCR

makes initial sample preparation easier and cheaper.
Futuristic methods of DNA Sequencing
http://web.virginia.edu/Heidi/chapter12/
chp12.html
http://www.sequencingmethods.com/d
na-sequencing-methods.html
http://www.genome.gov/27541189