Download 9 DNA Sequencing and Generation of Mutations

9
DNA Sequencing and Generation
of Mutations
9.1
DNA Sequencing Methods
1. The Sanger method: enzymatic
2. The Maxam-Gilbert method: chemical
Frederick Sanger
Walter Gilbert
Nobel price in Chemistry in 1980
3. Pyrosequencing
4. Nanopore sequencing
9.1.1
The Sanger Method
The Sanger Dideoxy-Mediated Chain
Termination Method
General Strategy for DNA Sequencing
Scheme: Sanger-Based Capillary
Electrophoresis Sequencing System
Each reaction contains a
different fluorescent tag
Sanger-Based Capillary
Electrophoresis Sequencing Systems
ƒ Up to 700 bases of sequence information from
each of 96 templates
ƒ 67,000 bases per h
ƒ 25 million bases in one four - hour run
9.1.2
The Maxam-Gilbert Method
Principle: Chemical degradation of the DNA
1. Chemical modification of the base
2. Elimination of the modified base
3. Cleavage of the DNA strand at the
location of the eliminated base
The sequencing reactions:
• C: hydrazine in sodium chloride cleaves the C
base only
• G: G bases are methylated at N4 by
dimethlysulfate and then cleaved with alkali
• G + A: glycosidic bond between base and
sugar destroyed by formic acid treatment
• T + C: hydrazine cleaves the pyrimidine ring
system
Final step: Piperidine treatment
Chemical Sequencing: The MaxamGilbert Technique
Autoradiogram of a Chemical
Sequencing Gel
9.1.3 Pyrosequencing
Principle:
Based on the pyrophosphate (PPi) released
during the DNA polymerase reaction
The Sequence of Reactions
M Ronaghi (1996) Anal. Biochem. 242: 84
Pyrosequencing
9.1.4
Nanopore sequencing
Principle:
Molecules carrying a net electric charge are
electrophoretically driven through a pore by an
applied electric potential and the conductance
is dependent on the nucleotide within the pore
DW Deamer (2006) Trends Biotechnol. 18: 147
M Rhee (2007) Trends Biotechnol. 25: 174
Nanopore Sequencing
1.3 nm
Sequencing of the human genome in 20 h
The α-Hemolysin Nanopore
Characteristics:
ƒ Monomeric, 33 kDa protein produced by S.
aureus
ƒ Monomers self-assemble as a heptamer on
synthetic lipid bilayers to form a 1.5 nm‚ diameter aqueous channel through the membrane
ƒ Pore remains open at neutral pH and high ionic
strength
ƒ Pore passes a steady ionic current in a
detectable range ensuring a low level
background electrical noice
Ribbon Representation of the αHemolysin Heptamer
cis
ssDNA:
13 nm
trans
D Branton (2008) Nature Biotechnol. 26: 1146
454 Life Sciences
DNA sequencer: € 500 000
Able to read >20 million bases during a 5-h-run
Readout:
24 nucleotides per sec at present
450 000 nucleotides in the future
Total Costs for Sequencing of the
Human Genome
2003
3 billion US$
2006
20 million US$
2009
100 000 US$
2014
The $1000 genome
The classical integrated sequencing
pipeline includes
ƒ Library construction
ƒ Template amplification
ƒ DNA sequencing
Is it possible to improve the sequencing
pipeline ?
Amplification of ssDNA
D MacLean (2009) Nature Rev Microbiol 7: 287
9.2
Sequencing of Whole Genomes
1. Clone-by-clone strategy
2. Shotgun sequencing
3. EST
Clone-by-Clone Strategy
1. Gene library of a bacterial chromosome, e.g.,
in a cosmid vector
2. Ordering the inserts around the chromosome
3. Subcloning of inserts as smaller fragments
4. DNA sequencing of the small fragments
Shotgun Sequencing
1. Shearing of the chromosomal DNA into about
1 kb fragments
2. Cloning of these fragments into M13
3. DNA sequencing (Sanger)
4. Assembling the DNA fragments together by
an appropriate computer program
Craig Venter
1995: Haemophilus influenzae
1.8 Mbp
EST = Expressed Sequence Tag
EST = cDNA clone library; most of the clones
contain only part of the genes
Craig Venter prepared ESTs from the human
brain and tried to get them patented
AAAA
cDNA
EST
9.3
Generation of Mutations in
Cloned DNA Fragments
1. Generation of small deletions
2. Generation of small insertions
3. Generation of point mutations
Isolation of Mutations: Theoretical
Considerations
1. Point mutations
ƒ At random: Chemical mutagenesis, UV
irradition, mutator strains; transposons
ƒ Conditional lethal mutations in essential genes:
- ts – mutations, - cs – mutations
- gene fused to a regulatable promoter
(depletion assay)
2. Knockouts
ƒ Insertion of a marker (antibiotic resistance gene)
ƒ Replacement of the whole gene by a marker
ƒ Gain-of-function mutation
ƒ Loss-of-function mutation
ƒ Synthetic lethality:
ΔdnaK : viable
Δtig : viable
ΔdnaK Δtig : lethal
9.3.1
Generation of Deletions
Possibilities:
1. Digest with restriction enzyme
leaving overhangs; remove these
overhangs; ligate the blunt ends
2. Remove internal fragment
3. Linearize within the gene; BAL31
digestion (exonuclease)
4. PCR
Generation of a Deletion in a Cloned
DNA Fragment
inverse
9.3.2
Generation of Insertions
Possibilities:
1. Cut with a restriction enzyme
leaving 5' overhangs; fill-in
2. Cut with restriction enzyme
leaving blunt-ends; addition of a
linker
Generation of Insertion and Deletion
Mutations by the Splice Overlap Extension
(SOE) Method
Insertion
Deletion
9.3.3
Generation of Point
Mutations with PCR
1. Single-priming method
Michael Smith
2. Double-priming method
3. Kunkel method
1932 – 2000
Canadian Biochemist
Nobel price in Chemistry in 1993
The Single-Priming Method
Double-Priming
Method Using two
Primers
mutS: no mismatch
repair
The Kunkel Method
dut: dUTPase
ung: uracil-DNA-glycosylase
TA Kunkel (1987) Methods Enzymol. 154: 367
Error-prone PCR
ƒ Taq polymerase (no proofreading)
ƒ Unequal dNTP concentration
ƒ High Mg2+ concentration
ƒ Partial replacement of Mg2+ by Mn2+
ƒ Incorporation of mutagenic dNTP
analogs (e.g., 8-oxo-dGTP)
Molecular diversity of enzymes can
be obtained by:
ƒ Targeted amplification of mutant strand
(TAMS)
ƒ DNA shuffling
ƒ Staggered extension
ƒ Incremental truncation for the creation
of hybrid enzymes (ITCHY)
Objective of these techniques:
ƒ To improve enzyme
- activity (pH-, temperature-optimum)
- stability
- folding
ƒ To alter substrate specificity
ƒ To combine useful mutations
Targeted Amplification of Mutant Strand
(TAMS): Introduction of Multiple Point
Mutations
( = primer)
T4 DNA polymerase
no 3'→ 5' exonucl.
T4 ligase
3' nucleotides of
PCR5' and PCR3'
match the mutant
nucleotide
L Young (2003) Nucl. Acids Res. 31: e11
DNA Shuffling
ƒ Powerful process for directed evolution
ƒ Generates diversity by recombination
ƒ Can be used to combine different
mutations
ƒ Introduced in 1994: WPC Stemmer
(1994) PNAS 91: 10747
WPC Stemmer (1994) Nature 370: 389
JE Ness (1999) Nature Biotechnol. 17: 893
Principle of DNA Shuffling
⇓
DNase I, Mn 2+
⇓ de- and renaturation
⇓ T4 DNAP
DNA Shuffling
A Crameri (1998) Nature 391: 288
Staggered Extension
⇓
⇓ de-, renaturation
⇓
H Zhao (1998) Nat. Biotechnol. 16: 258
Incremental Truncation for the
Creation of Hybrid Enzymes (ITCHY)
Combinatorial strategy for creating hybrids
between genes that lack DNA homology
Idea: To mix functional domains of two proteins
M Ostermeier (1999) PNAS 96: 3562
M Ostermeier (1999) Nature Biotechn. 17: 1205
S Lutz (2001) Nucleic Acids Res. 29: e16
ITCHY
Combinatorial
Protein Engineering
1. Cloning of fragments of genes
A and B into two compatible
vectors
2. Linearization of the vectors
3. Exonuclease III (3'→5') + S1
4. Klenow (end repair); NsiI of
one vector
5. Ligation of the two plasmids
6. Transformation
7. Selection or screening