Introduction to DNA Sequencing

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Introduction to DNA Sequencing
2. Determine which base terminates each fragment
3. Order the fragments
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Maxam and Gilbert Sequencing
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Maxam and Gilbert Sequencing
Reaction 1 Dimethylsulfate (alkaline) cuts
G Reaction 2 Dimethylsulfate (acidic) cuts G and
A Reaction 3 Hydrazine (piperdine) cuts C and
T Reaction 4 Hydrazine (2M NaCl) cuts C
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Maxam and Gilbert Summary
Advantage No primer is required
Disadvantages Requires single-stranded DNA
One label - poor sensitivity Nasty chemical
method
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Dideoxy Sequencing
1. 5 Phosphate Reacted with 3 Hydroxyl
2. What if no 3 Hydroxyl?
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bio401/DNASeq.html
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Sanger Sequencing
1. Dideoxy G, labeled C, 4 dNTPs
2. Dideoxy A, labeled C, 4 dNTPs
3. Dideoxy T, labeled C, 4 dNTPs
4. Dideoxy C, labeled C, 4 dNTPs
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This figure was taken from lthttp//www.plattsburgh
.edu/acadvp/artsci/biology/bio401/DNASeq.htmlgt
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Improved Dideoxy Sequencing
A
C
G
T
GACT CTGA
G
A
C
T
Generate Nested Sets for G,A,C,T in One Reaction
1. Must detect each dideoxy nucleotide uniquely
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DyeDeoxy Terminators
The dideoxy nucleotides are labeled with
different fluorophores that are spectrally
distinct - i.e. they emit different colored light
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Prokaryotic Gene RegulationThe Operon System
Regulatory Elements
Gene 1 Gene 2 Gene 3
Gene 4
One Set of Regulatory Elements Controls Multiple
Genes mRNA is PolyCistronic - codes for
multiple proteins
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Prokaryotic Gene Structure
Promoter
Operator
RBS ATG
Stop
Promoter Operator Ribosome Binding
Site ATG Stop
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Lac Operon
Promoter Operator
lac Z (4100 bp) lac y (900 bp) lac
a (900 bp)
b-Galactosidase Permease
Transacetylase
Promoter Operator
lac i (1200 bp)
lac Repressor
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Promoter Sequence SetsTranscription Direction
RNA Polymerase
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3
TTGACA-----TATAAT
3
5
AACTGT-----ATATTA
-35 -10
RNA Polymerase
5
3
ATTATA-----TGTCAA
3
5
TAATAT-----ACAGTT
-10 -35
Antiparallel Chemical Direction of DNA
Allows Direction of RNA Polymerase To Be Encoded
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Lac Operon
lac y lac a
lac y lac a
lac y lac a
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Lac Operon
Or IPTG
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Lac Operon Induction
Lactose Galactose Glucose
Quantity
Time
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Molecular Biology Details
1. Isnt the pUC 18 lacZ gene too short?
2. Doesnt E. coli have its own lac operon?
3. Is IPTG necessary to get blue colonies with
pUC?
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LacZ in pUC
1. lacZ in pUC is too small, doesnt code
for full enzyme 2. pUC lacZ fragment is
a-peptide
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Blue-White Color Test
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Engineered E. coli
Engineered E. coli
1. Remove native lacZ 2. Replace with plasmid
containing mutated lacZ
F Episome
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Trans-Complementation
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Inserts Disrupt lacZ
HindIII
EcoR1
No Insert Blue Colony
Insert White Colony
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Lac Operon
lac y lac a
lac y lac a
lac y lac a
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IPTG Induction
IPTG doesnt help with pUC system
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Blue - No Insert
Light Blue - Insert, or other alteration
White - Insert, or other alteration
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Protein Expression
Maximize Protein Production 1. Optimize
Transcription/Translation
Regulate Timing of Expression 1. All
cellular resources to target 2. Toxic
proteins ok
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Protein Production
Folding
Stability
Protease Stability
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Bacterial Cell Structure
Outer Membrane
Periplasm
Cytoplasm
Cytoplasmic Membrane
Two Compartments Cytoplasm Periplasm
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Compartmental Properties
Cytoplasm
Redox potential is lower than S-S bonds No
disulfide bonds in cytoplasm Proteases
present Proteins needing disulfides for
folding cant be made
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Compartmental Properties
Periplasm
Redox potential is higher than S-S bonds
Disulfide bonds can form in periplasm
Proteases present Proteins needing disulfides
for folding can be made
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Cytoplasmic Expression
Engineered E. coli
Promoter/Op
lacUV5
T7 Polymerase Gene
IPTG T7 Pol On
Lots of Insoluble Aggregated Protein Inclusion
Bodies
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Inclusion Bodies
Periplasmic
Cytoplasmic
http//web.mit.edu/king-lab/www/research/Scott/Sco
tt-Research.html
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Protein Transport
Outer Membrane and Periplasmic Proteins
Promoter
RBS
ATG
Coding Sequence
Leader Peptide (21 a.a.)
Peptidase
Periplasm
Cytoplasmic Membrane
Leader Peptide
Cytoplasm
Ribosome
mRNA
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Compartmental Targeting
Promoter
RBS
ATG
Target Protein, e.g. antibody
Leader Peptide (21 a.a.)
Antibody folds In periplasm
S-S
S-S
Periplasm
Cytoplasmic Membrane
Cytoplasm
Antibody unfolded In cytoplasm
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T7 Expression System
Extremely High Activity (Processivity)
Super Polymerase
High Specificity 1. T7 doesnt recognize
E. coli promoter 2. E. coli doesnt
recognize T7 promoter
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The pET System
IPTG Regulates T7 Polymerase Expression
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pET Plasmids
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T7 Summary
Chromosome
E. coli BL21(DE3)
T7 Pol Gene
T7 Pol
pET
Target Gene
T7 Promoter
X
E. coli Pol
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E. coli Growth Curve
Stationary Phase
Death Phase
IPTG
Cell Density
Log Phase
Lag Phase
Time