DNA

1. DNA Sequencing
Basic Principles

2. Basic Principles
 All current practical DNA sequencing techniques can be divided into
four major steps:
1. Labeling of DNA so that small quantities can be easily detected,
traditionally done by labeling with either P32 or S35
2. Generation of fragments for which the specific bases at the 3’ end
are known
3. Separation of fragments using gel electrophoresis sensitive enough to
resolve differenced in size of one nucleotide
4. Fragment detection

3. Sanger Sequencing
 The Sanger sequencing method takes advantage of the way that
normal DNA replication occurs
 For DNA to be extended using normal DNA polymerases, a
hydroxyl group must be present at the 3’ carbon on deoxyribose
 Fragments are generated by spiking reactions with small quantities 2’
3’ dideoxy nucleotides which terminate polymerization whenever
they are incorporated into DNA
 Polymerases used must lack 3’ to 5’ exonuclease proof reading
activity for this method to work

4. H
P
O
OH
OH
HO
O
O
CH2
NH2
N
N N
N
Sugar
Base
Phosphate
3’
5’
2’
1’
4’
Dideoxynucleotides
 DNA Sequencing using the Sanger
method involves the use of 2’3’-
dideoxynucleotide triphosphates in
addition to regular 2’-deoxynucleotide
triphosphates
 Because 2’3’-dideoxynucleotide
triphosphates lack a 3’ hydroxyl group,
and DNA polymerization occurs only
in the 3’ direction, once 2’3’-
dideoxynucleotide triphosphates are
incorporated, primer extension stops
H
2’3’-dideoxynucleotide
monophosphate
2’-dideoxynucleotide
monophosphate

5. H
P
O
HO
O
O
CH2
H
OH
P
O
O
HO
O
O
CH2
H
P
O
OH
HO
O
O
CH2
NH2
N
N
N
N
O
O
NH2
N
NH
N
N
N O
NH2
N
2’3’
dideoxy-
nucleotides
Terminate
DNA
Replicaton O
H
P
O
HO
O
O
CH2
HO
H
H OH
P
O
OH
O
O
CH2
OH
H
P HO
O
O
CH2
H
O
H2O

6. DNA Sequencing
Plasmid (or phage)
with cloned DNA
fragment
Primer Binding sites
Cloned
fragment
Primer

7. AGCTTAGC TGCAATGC 3’
Region to be sequenced Primer annealing site
5’
3’ACGTTACG 5’
Add primer
3’-A*TGCACGTTACG-5’
AGCTTAGC TGCAATGC-3’
5’
Labelling &
termination
Add [ 35S]dATP,dTTP,dGTP,dCTP+DNA Pol (Synthesis
proceeds until dNTPs are exhausted)
Divide into four rxns
+ddATP + 4dNTPs +ddTTP + 4dNTPs +ddGTP + 4dNTPs ddCTP + 4dNTPs
Run a gel in four separate lanes
SANGER’S DIDEOXY SEQUENCING

8. 5’
5’
GCTAAGC
‘A’ reaction ‘G’ reaction
‘T’ reaction ‘C’ reaction
5’AGCTTAGC3’
GCTA 5’
5’
5’
5’
5’
5’
GCTAA
GCT
GCTAAGCT
G
GCTAAG
GC
GCTA
GCTAA
GCT
5’GCTAAGCT3’
G
GCTAAG
GC
GCTAAGC
A T G C
5’CTAAGCT 3’
SANGER’S DIDEOXY SEQUENCING ( contd.)

9. Pros and Cons of the Sanger Method
 It is more amenable to automation
 Fewer dangerous chemicals are used, but acrylamide and
P32 or S35 are still a problem
 Gels or autorads are generally cleaner looking and the
reading of bases is lot easier. But casting gel is a bottleneck
 The bottom line: Without improvements in automation,
detection and separation technologies Sanger sequencing is
still very labor intensive

10. Sequencing Method Refinements
 Because of difficulties intrinsic to the Maxam-Gilbert
chemical sequencing strategy, efforts at improvement have
been concentrated on the Sanger method
 Major improvements in the following areas have been
achieved
 Labeling and detection
 Fragment separation
 DNA Polymerases used in sequencing and resulting
strategies for generation of fragments
 Automation

11. CYCLE SEQUENCING
ddA+ddT+ddG+ddC+
Taq POL+ dNTP
+
Reaction mix
Denaturation
Original
Template
Extension
A
AC
ACC
ACCG
ACCGT
ACCGTAT
ACCGTA
Annealing
ADVANTAGES
•A broader range of starting DNA quantity
• High temp. reduces 2ndary structures of template
• High temp. reduces non-specific extension
• No alkali denaturation for ds DNA
• Easy to perform
primer
+

12. Labeling and Detection
 The most significant advance in labeling has been the
production of electrophoretically neutral dyes that
fluoresce at specific wavelengths when excited by laser
produced light over a very narrow range of wavelengths
 These dyes, when attached to primers allow detection
down to 15 attomoles (10-18)
 That’s less than 107 molecules!

13. Applied Biosystems
 Applied Biosystems (ABI) has developed fluorescent dye
systems further and improved methods for loading and
electrophoresis
 Four dyes each of which fluoresce at a different
wavelength, but having about the same impact on
electrophoritic mobility can be used to label either primers
or the nucleotides that terminate a reaction
 If terminator dyes are used, the entire sequencing reaction
is reduced to one tube from 4 in conventional Sanger
sequencing
 Instead of polyacrylamide slab gels, a single capillary can
be used with a liquid polymer that is replaced after each
individual run

14. A
AC
ACC
ACCG
ACCGT
ACCGTAT
ACCGTA
ACCGTA
A
C
G
T
ENZ.dNTPs
ddNTPs
A
ACCGTAT
ACCG
ACC
AC
ACCGT
DYE TERMINATOR LABELLING
ddA
ddC
ddC
ddG
ddT
ddT
ddA
DYE PRIMER LABELLING
Template
+
Primer
Annealing Extension

15. COMPARISON BETWN DYE TERMINATOR & DYE PRIMER
DYE TERMINATOR DYE PRIMER
• Unlabelled primer labeled primer
• Single tube rxn. Four tube rxn
• False stops go un noticed could be a problem
• Single lane run Single lane after pooling
• Peak heights are uneven peak heights are even
• Lesser read length longer read length

16. …..
Heat plate
Liquid
polymer
ATTGC A
ABI Prism 310 System
Laser
Beam
splitter
Detectors
-
+
Window
Sequencing
reaction
Sequencing
reaction
Capillary

17. The State of the Art
 The ABI Prism 310 (1 capillary), 3100 (16
capillaries) and 3730xl (96 capillaries) represent the
current state of the art in automated sequencing
machines
 The 3100 can run up to 184,000 bases per day
 The 3730xl can run up to 1,104,000 bases per day
 Large sequencing facilities, like Sanger Centre,
have full of these machines which can run 24 hours
a day with very little down time for routine
maintenance

18. 3730/3730xl System Hardware
Piercing Station
Plate Stacker
Polymer Delivery System
Autosampler
Internal Bar Code Reader

19. Next Generation Sequencing Technologies

20. ~600 genes
of interest
~200 lung
adenocarcinoma
samples
• Sequencing Centers: BCM-HGSC, BI, WUGSC
• Cancer Centers: MSKCC, DFCI, SCC, MDA
Tumor Sequencing Project

21. Amplify to form clusters
Sequence
one base at a time
T
A C
G
Lazer
Linker ligated DNA
Solexa sequencing

22. ©2001 Timothy G. Standish

23. 454
sequencing
Margulies M et al.
Nature. 2005 Sep
15;437(7057):326-7.

24. 454
sequencing
24
Margulies M et al.
Nature. 2005 Sep
15;437(7057):326-7.

25. ©2001 Timothy G. Standish
Applications of Next Generation DNA Sequencing
De Novo sequencing
Targeted sequencing/Exome sequencing
Whole Genome re-sequencing
Chromatin Immunoprecipitation sequencing (ChIP-sequencing)
Methylome analysis
Transcriptome analysis
Small RNA analysis

26. ©2001 Timothy G. Standish
Loss of tumor
suppressor
function
Gain of oncogenic
function
Mutant p53 – a two faced gene
1.00
0.96
0.86
0.72 0.73
0.22
0.00
0.20
0.40
0.60
0.80
1.00
Mock Sc siRNA EFNB2 siRNA
VIABILITY
-5FU +5FU
1.00
0.95
0.65
0.76
0.66
0.26
0.00
0.20
0.40
0.60
0.80
1.00
Mock Sc siRNA UBE2C siRNA
VIABILITY
-5FU +5FU
By candidate approach
ChIP-Seq
Why should I initiate my PhD project from whole genome analysis?

27. ©2001 Timothy G. Standish
27
Alternatives to Dye Terminator Sequencing
 454 Sequencing is a massively-parallel sequencing-by-synthesis (SBS) system
 capable of sequencing roughly 20 megabases (20,000,000 bp) of raw DNA sequence per 4.5-
hour run
 Compare to best dye terminator sequencing rig today :ABI 3730xl
– (192 capillaries x ~1000 bp) in 5 hrs (2 2.5 hr runs) = 196,000 bp
 454 sequencing relies on fixing nebulized and adapter-ligated DNA fragments to small DNA-
capture beads in a water-in-oil emulsion.
 DNA is fixed to these beads is then amplified by PCR.
 Each DNA-bound bead is placed into a ~44 μm well on a PicoTiterPlate, a fiber optic chip. A
mix of enzymes such as polymerase, ATP sulfurylase, and luciferase are also packed into the
well.
 The four nucleotides (TAGC) are washed in series over the PicoTiterPlate.
 If a nucleotide complementary to the template strand flows into a well, the polymerase
extends the existing DNA strand by adding nucleotide(s).
 Addition in a reaction that generates a light signal that is recorded by acamera in the
instrument.

28. Future
$ 1000 a Human Genome (3000Mb)

29. Genome size 3000 Mb
Req'd coverage 6 12 20
3730 454 FLX Solexa
bp/read 600 250 32
Reads/run 96 400,000 28,000,000
bp/run 57,600 100,000,000 896,000,000
#/runs req'd 312,500 360 67
Cost per run 48
$ 6,800
$ 9,300
$
Total cost 15,000,000
$ 2,448,000
$ 622,768
$
Present