DNA Sequencing

1
Lecture 8 DNA Sequencing Introduction Reminder
of nucleotide chemistry Chain termination
sequencing (Sanger-Coulson) Automated
sequencing Genome sequencing importance and
strategies New sequencing technologies
Mutagenesis of cloned genes Introduction Random
mutagenesis Site directed mutagenesis Protein
engineering
2
Learning outcomes After this lecture you
should be able to describe the use and
methodology of small and large scale DNA
sequencing describe the use and methodology
of random and site directed mutagenesis
3
DNA sequencing Goal to determine the order of
nucleotides in a piece of DNA DNA sequence gives
a wealth of information Amino acid sequence
Homology to proteins in other organisms,
elucidate function Promoter structure Intron/Exo
n structure Mutations (diagnosis of genetic
diseases) Evolutionary relationship between
organisms
4
Principle of DNA sequencing Use a primer and
polymerase to copy single stranded DNA
molecule Detect the order in which the different
nucleotides are added to the newly synthesised
strand
sequencing primer
Polymerase
Single stranded DNA molecule
5
Nucleotides are the building blocks of DNA and RNA
5'
5'
CH2
A/C/T/G
P
P
P
CH2
A/C/U/G
P
P
P
O
O
C
C
1'
4'
C
C
1'
4'
C
C
2'
3'
C
C
2'
3'
OH
H
OH
OH
RNA Ribo Nucleic Acid
DNA Deoxy ribo Nucleic Acid
phosphate group
P
6
DNA is a polymer of nucleotides
5'
CH2
A/C/T/G
P
A chain of dNTP's DNA
O
C
C
C
C
O
H
CH2
A/C/T/G
P
O
C
C
C
C
A single dNTP
5'
O
H
CH2
A/C/T/G
P
P
P
O
CH2
A/C/T/G
P
O
C
C
1'
4'
C
C
C
C
2'
3'
3'
C
C
OH
H
O
H
7
Nucleotides need a 3' Hydroxyl and 5' Phosphate
group to polymerise
P
P
P
CH2
A/C/T/G
CH2
A/C/T/G
P
P
P
O
O
C
C
C
C
C
C
C
C
OH
H
O
H
P
CH2
A/C/T/G
P
P
P
CH2
A/C/T/G
O
O
C
C
C
C
C
C
C
C
OH
H
OH
H
8
Sanger-Coulson sequencing the use of chain
terminating nucleotides
5'
5'
CH2
A/C/T/G
P
P
P
CH2
A/C/T/G
P
P
P
O
O
C
C
1'
4'
C
C
1'
4'
C
C
2'
3'
C
C
2'
3'
OH
H
H
H
Deoxy NTP (dNTP)
Dideoxy NTP (ddNTP) Terminates DNA chain
9
Principle of Sanger-Coulson sequencing synthesize
DNA in the presence of dNTP's AND ONE
ddNTP Polymerise in presence of dATP dTTP dCTP
dGTP ddATP Terminates chain!!!!
Radioactive
cgtatacgcgtatcgctatcgcgtagtactcgcgtca
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Principle of Sanger-Coulson sequencing Run 4
separate reactions with different
chain-terminating nucleotides
cgtatacgcgtatcgctatcgcgtagtactcgcgtca
dNTP's ddATP
dNTP's ddTTP
dNTP's ddCTP
dNTP's ddGTP
11
Principle of Sanger-Coulson sequencing Run
mixtures on separate lanes on polyacrylamide
gel Expose to X-ray film
A
T
C
G
cgtatacgcgtatcgctatcgcgtagtactcgcgtca
dNTP's ddATP
dNTP's ddTTP
Migration
dNTP's ddCTP
dNTP's ddGTP
cgtatacgcgddT
cgtatacgcddG
cgtatacgddC
cgtatacddG
cgtataddC
One sequencing reaction reads 400 bases
12
Automated DNA sequencing using the Sanger-Coulson
method Use fluorescent ddNTP's of different
colours, reaction can be performed in single
tube Separate DNA fragments through capillary
electrophoresis
cgtatacgcgtatcgctatcgcgtagtactcgcgtca
dNTP's ddATP
Migration
dNTP's ddTTP
dNTP's ddCTP
cgtatacgcgddT
dNTP's ddGTP
cgtatacgcddG
cgtatacgddC
cgtatacddG
cgtataddC
Detector
One sequencing reaction reads 750 bases
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Example of automated sequencing output
14
Genome sequencing Sequence all the DNA from a
single organism and make it available in a public
database Includes coding and non-coding
DNA Greatly facilitates research, allows in
silico hybridisation, primer design, etc.
15
Genome sequencing Species Genome size (Mb)
of 750 bp sequencing runs needed E.
coli 4.6 6'133 S. cerevisiae 12.1 16'13
3 C. elegans 97 129'333 D.
melanogaster 180 240'000 H.
sapiens 3200 4'266'667 Need for highly
efficient automated sequencing approach!
Robots perform the sequencing reactions
Automated sequencers read the sequences
Computers Analyse the data
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First step in genome sequencing creating a
genomic library Break down genome using partial
restriction digestion or sonication (sonication
is more random) Select for specific
size Ligate into suitable vector Transform and
order in microwell plates
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Two alternative sequencing approaches Shotgun
approach and clone contig approach Contig set
of overlapping DNA sequences
Clone contig Determine overlap
Disadvantage laborious
Sequence overlapping clones
Shotgun Sequence randomly
Disadvantage Closing gaps requires high level of
redundant sequencing
Continuous DNA sequence
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Clone contig approach How to assemble a clone
contig? Example chromosome walking
A
B
C
D
E
F
G
H
I
J
K
L
M
N
1
2
A1
3
F4
N3
4
5
6
7
8
Probe with clone A1
Use F4 and N3 as probe
But this is slow.........
Keep on walking! Use F4 and N3 as probes
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Clone contig approach How to assemble a clone
contig? Example clone fingerprinting Cut
different clones with restriction enzyme and look
for same-sized bands
1
2
3
identical size clones overlap
identically sized bands
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New sequencing technologies 454 Life Sciences
developed machine that can sequence 96 MB per
day, would only take 33 days to sequence human
genome (comparison Human Genome Project took 13
years.....)!!!!! Even faster technologies are
currently being developed Near future fast and
cheap technology allows sequencing of individual
human genomes within days Huge implications for
medicine and concerns for privacy For
overview of new technologies Science Vol 311
(March 17 2006) page 1544
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Mutagenesis Goals to change the expression or
function of a protein to study protein
structure-function relationships Random
mutagenesis randomly mutagenise a gene Site
directed mutagenesis introduce a specific
mutation in the DNA
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Random mutagenesis the use of error prone
PCR Use a special error prone DNA polymerase,
e.g. Mutazyme II (Stratagene). Between 1 and 16
mutations per kb Mutation rate controlled by
varying the number of amplification cycles
23
Site directed mutagenesis introduce mutation
through primer Allows predictable and precise
deletion, insertion or change of single or
multiple nucleotides
primer
deletion
template
T
G
insertion
G
change
T
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QuickChange Site Directed Mutagenesis
Many bacterial strains methylate vector DNA,
PCR-amplified DNA is not methylated
X
X
X
X
X
not methylated
hemi methylated
15 PCR cycles Denature Anneal Extend
fully methylated
X
DpnI digestion Only digests methylated
and hemi-methylated DNA
X
not methylated
25
Protein engineering design or change a protein
to improve it's properties e.g increase of
enzyme activity or protein stability
subtilisin Rational design Design a protein
change on the computer Requires thorough
understanding of protein folding, structure and
function With current level of understanding it
is very difficult to predict a beneficial change
to a protein Directed evolution Randomly
mutagenise a protein and test mutants for
improved function/stability
26
Protein engineering through directed evolution
transfer to microtiter plates Express protein
mutagenise transform
Test individual clones for improved properties e.g
. heat resistance
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So far...... Lecture 4/5 Introduction to the
use of cloning tools Lecture 6 How these tools
are used to create a DNA library Lecture 7 How
DNA libraries are used to find your favourite
gene/protein How PCR can be used to amplify DNA
Lecture 8 Sequencing and mutagenesis of cloned
genes Lecture 9 Expression of medically and
commercially important eukaryotic proteins in E.
coli