solexa aka: sequencing by synthesis 2) attach each adaptor to a flow cell to create ssdna templates....
TRANSCRIPT
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SOLEXA aka: Sequencing by Synthesis
2) Attach each adaptor to a flow cellto create ssDNA templates. Aftereach round of amplification, the strands are denatured to createmore templates.
1) Randomly cut DNA fragments and ligateadaptors to both ends
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3) Add four labeled reversible terminators, primers, and DNA polymerase.
4) Wash off all of the unbound components, excite the laser, and read the emitted fluorescence of the first base for each cluster.
5) The blocked 3’ terminus and fluorophore has to be removed before repeating to determine the next base
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434 Sequencing
• Capable of sequencing 400-600 megabases of DNA in a 10 hour run
• Applications:1.Whole genome sequencing2.Amplicon sequencing3.Transcriptome sequencing4.Metagenomics
• Advantages: low cost, longer reads and higher accuracy than the Sanger chain-termination method
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434 SequencingConcept
• Large-scale parallel pyrosequencing, “sequencing by synthesis”:
1.Each nucleotide is added in turn, producing PPi
2.Sulfurylase uses PPi to produce ATP, that is used by luciferase to produce light
3.Apyrase denatures the remaining dNTP at each step
4.The light produced with the addition of each nucleotide can be plotted to form a sequence
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434 SequencingTechnology
• DNA is fractionated, 5’ biotin labeled and attached to streptavidin coated beads. The non-labeled strand is released.
• The DNA on each bead is amplified onto the bead
• One bead is deposited in each well
• Pyrosequencing takes place in the Genome Sequencer FLX instrument
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Applied Biosystems SOLiD System
© Copyright 2008 Applied Biosystems. All Rights Reserved.
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© Copyright 2008 Applied Biosystems. All Rights Reserved.
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* The position on the DNA at which replication start points are found. (4 methods discussed)
* A DNA sequence that when added to a non-replicating DNA causes it to replicate.
* A DNA sequence whose mutation abolishes replication.
* A DNA sequence that in vitro is the binding target for enzyme complexes known to function in initiation of DNA replication.
Characteristics used to define Origins of Replication
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1) In synchronously replicating cells, density transfer experiments can reveal which sequences replicate earliest. What is the direction of fork
movement here?
Time
% o
f p
rob
e in
HL
DN
A0
100
A B C
C B A
Genes on achromosome
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TR50
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8
Hr of S phase
% c
um
ula
tiv
e r
ep
lic
ati
on
Gene A
Gene B
TR50 - Time at which 50% of the locus is replicated
In the example below,‘probe A’ has a TR50 of 1.25hr (80% at 2hr, 0% at 0hr)‘probe B’ has a TR50 of 6.33hr (100% at 8hr, 40% at 6hr)
TR50 Calculation for human chromosomal probes on genome tiling arrays (ENCODE project)
ProbeProbe
Time Point Probe A Probe B
0-2 .80 .10
2-4 .10 .10
4-6 .10 .20
6-8 0 .60
Example:
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Predicted origins, UCSC genome browser, ENCODE: UVa DNA replication tracks
Neerja KarnaniChris TaylorAnkit Malhotra
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EcoRIBamHI
BamHI EcoRI
2) Electron microscopy of bubbles after restrictionenzyme digestion
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3) Hybridizing lagging strand nascent DNA to strand-specific fragmentsshows site of switch from leading to lagging strand synthesis
A B C DUpper strand fragments
Lower strand fragments
E F G H
Nascent DNA used to probe DNA fragments:
A B C D
E F G H
A B C D
E F G H
DNA spotted on membrane
Autorad after hybridizing nascent strands
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3b) Hybridizing of leading strand nascent DNA shows sites near origins that are enriched in nascent strand
A B C DProbes E FG H
Nascent DNA 500-2000 base long used to probe fragments along chromosome
DNA spotted on arrayNascent strand peak at origin
Okazaki fragments: <500 bases longLeading strand nascent strands: > 500 bases longLeading strand nascent strands near origins < 2000 bases long
A B C DE F G H
A B C DE F G H
Nas
cent
str
and
abun
danc
e
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4) 2D Gel electrophoresis of replication intermediates followed byhybridization with a DNA fragment reveals whether a replication
bubble originates in the fragment
Dimension 1: separates by size; dimension 2: separates by shape
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Sequence that when added to a non-replicating DNA causes it toreplicate; this approach was used to identify
Autonomously Replicating Sequences (ARS) in yeast.
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Sequence that when mutated causes a replicating DNA tofail to replicate; this approach was used to identify
essential elements in an (ARS) in yeast
Yeast containing Leu2 on a plasmid
Plate yeast on Leucine minus plates to estimate rate of plasmid loss
Grow under non-selective conditions
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Linker scanning mutations in yeast ARS1
% U
RA
+ c
olo
nie
sExamples of mutants
AB1B2B3WT-ARS1
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OriC in E. coli chromosomal DNA
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Initiation of DNA replication in E. coli
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Nature Structural & Molecular Biology – 13, 676 - 683 (2006) Berger JM
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Two primosomes for E. coli chromosomal replication
Site of primosome assembly oriC, A-site n'-pas, D-loop, R-loop
Recognition DnaA protein PriA protein
Auxilliary proteins for (HU for oriC) PriBloading helicase PriC
DnaT
Helicase DnaB/DnaC
Priming Primase
DnaA and oriC independent replication initiation in E. colifrom hairpins, D-loops and R loops
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SV40: a polyomavirus that is very useful forstudying eukaryotic replication enzymes
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Early genes : proteins essential for viral DNA replication
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T antigen binding sites cluster around the ori
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* 98% nuclear: NLS * origin-specific DNA binding * DNA independent ATPase * ATP dependent DNA helicase * Binds to DNA polymerases, AP2, p53, Rb * Undergoes phosphorylation etc.
Properties of SV40 T antigen
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COS cells (infected with SV40)(expresses T Ag)
+ plasmid + dNTP + rNTP+ 32PdATP
37o C, 60 min
Analyze products by electrophoresis and autoradiography
SV40 ori
SV40 - + ori - +
Li & KellySV40 DNA replicationIn vitro.PNAS (1984) 81:6973
What else could they doto validate their system?
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Fractionation to homogeneity
COS cells (infected with SV40)(expresses T Ag)
T Antigen frombaculovirus expression system
Human HeLa or 293 cells
cell lysate
PC
0.2 M KCl 0.66 M KCl
QS
0.2 M KCl 0.4 M KCl (PCNA)
ssDNA
0.6 M KCl 1 M KClRPA
+
(RF-C, pol + pol )
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Prokaryotic Function Eukaryotic
SSB ss DNA coating RPA
PRIMASE RNA primer synth. Primase subunitof DNA POL ,
5'-3' polymerase DNA POL ,DNA POL
POL III CORE 3'-5' exonuclease DNA POL 5'-3' exonuclease Fen1
COMPLEX ATP dependentclamploader
RF-C
CLAMP processivity factor PCNA
LIGASE seal nicks Lig1
DnaB Helicase ?
ELONGATION FACTORS ARE CONSERVED
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Subunit
Gene Bacterial Function Eukaryotic
dnaE | 5'-3'polymerase
DNA POL
dnaQ(mutD)
| POL IIICORE
3'-5'exonuclease
DNA POL
| 5'-3'exonuclease
Fen1
dnaX
dnaX | | ATP' |
COMPLEXdependentclamploader
RF-C
| |
dnaN CLAMP processivityfactor
PCNA
CONSERVATION FROM PROKARYOTES TOEUKARYOTES
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RF-C is a five-subunit complexAll subunits are related in sequence and have ATP binding motifsATP hydrolysis by RF-C is associated with the loading of PCNARF-C is the functional homolog of the clamp-loader complex
RF-C
RF-C
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Polymerase switching occurs even on lagging strands; pol does most of DNA synthesis
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Dna2 endonuclease is also necessary for this step
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How do you think the PCNA is removed after ligation?
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PCNA interacts with RF-C, pol , Fen1, DNA ligase,CAF1 and MCMT
Several of these have a common motifused in the interaction: Q-X-X-L/I/M-X-X-F-F/Y
p21/CIP1/WAF1, a protein induced by the tumor suppressor p53 uses the same motifto interact with PCNA
What effect is p21 expected to have on DNA replication/repair?
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FEN1 interacts with PCNA
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DNA Helicase
* Helicase binds ss DNA
* Hydrolyzes ATP to move alongssDNA and peel of substrate DNA
* Can move 5‘ to 3’ or 3‘to 5’ or in both directions on ssDNAand is classified as such
* Dimers or hexamers
P P
5‘ 3‘
3‘ 5‘ 5‘3‘
Substra
te
Boiled su
bstra
te
+he
licas
e
+helic
ase+
ATP
15mer 30mer
15mer30mer
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Conserved helicase motifs:
Includes the following for ATPbinding and hydrolysisI = Walker A motif GxGxGKTII = Walker B motif DEad
Binds ATP
Tight conformation
Hydrolyzes ATP
Relaxed conformation
Helicases are ATP driven molecular motors
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Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes)
Yeast containing Leu2 on a plasmid
Plate yeast on Leucine minus plates to estimate rate of plasmid loss
Grow under non-selective conditions
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MCM8
MCM9
Form hexamer
Associates with MCM2-7
Newbies of unknown function
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MCM homolog fromM. thermoautotrophicum(an archaebacterium)has helicase activity.
What direction?
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Mt MCM formsdouble hexamers
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T antigen helicase domain: XS Chen, Nature, 2003;423:512-8
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MtMCM-N terminal (non-helicase) domain: XS Chen, Nature Str. Biol. 2003;10:160-7
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Model of replicative double-hexameric helicase action : XS Chen, Nature, 2003;423:512-8