Automated Forward and Reverse Ratcheting of DNA In
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Transcript of Automated Forward and Reverse Ratcheting of DNA In
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AUTOMATED FORWARD AND
REVERSE RATCHETING OF DNA IN
A NANOPORE AT 5-A PRECISIONGerald M Cherf et al.
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GROUP MEMBERS
Pranav Varma
Nihal Sangeeth
Ghanim FajishAlex Johny
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INTRODUCTION TO NANOPORE SEQUENCING
The motive is to find the order in whichnucleotides occur in a strand of DNA.
The theory of nanopore sequencing is that when a
nanopore is immersed in a conducting fluid and a
potential is applied across it,an electric current due to conduction of ions
through the nanopore can be observed.
The amount of current is very sensitive to the size
and shape of the nanopore.
The current varies depending on whether the pore is blocked by a A,G,T or
C.
If single nucleotides (bases), pass through the pore this can create a
characteristic change in the magnitude of the current observed
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COMMON NANOPORE : ALPHA HAEMOLYSIN Alpha haemolysin (HL), from bacteria that
causes lysis of red blood cells, has beenstudied for over 15 years.
Studies have shown that all four bases can be
identified using ionic current measured across
the HL pore.
The structure of HL is advantageous to
identify specific bases moving through the
pore. The HL pore is ~10nm long, with two
distinct 5 nm sections.
The upper section consists of a larger,vestibule-like structure and the lower section
consists of three possible recognition sites (R1,
R2, R3), and is able to discriminate between
each base.
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6 FEATURES
Automated capture and processing of genomic DNAtemplates in single file order from a heterogeneousmixture over many hours
Systematic spatial control Temporal control
Absence of complex active voltage control
A sensor to identify single bases
Counter to identify nucleotides in homopolymericregions
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ENZYME MOTOR COUPLING
Means to slow down template strandmovement(average rate-~3 s per nucleotide @ 120
mV)(required rate-~0.1-1000 ms/nt)
Advantages
Systematic enzyme driven movement relative
to nanopore at milliseconds per nucleotide
Pull of the enzyme motor and force of electric
opposite to it helps hold the strand taut
Thereby base read errors due to brownian
motion are reduced
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DNAP AS ENZYME MOTOR COUPLES
T7 DNAP binds to ssDNA;catalyses ntadditions that advances template strandthrough the pore(applied voltage-80mV)
Use of T7 DNAP in sequencing not practicalfor 2 reasons
i. At most 3 sequential ionic current steps observedbefore t7 DNAP dissociation from template
ii. To remove blocking oligomer and bind t7 DNAP ,theDNA was tethered in pore and driven back and forthby reversing polarity at 10 ms intervals resulting inhigh crosstalk between pores
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DN P S ENZYME MOTOR COUPLES
Experimental data suggest phi29(a B-familypolymerase) bounds to DNA ~10000 times longerthan A-family polymerases(T7 DNAP)
Controlled sequential movement of atleast 50base through nanopore from a precise startingpoint in a primer strand without active voltagecontrol
Rate of elongation and template displacementwas tens of milliseconds/nt
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BLOCKING OLIGOMER
WHY? Prevent replication,elongation and excision in bulk phase
For capture activation and electrophoresis of upto 500 DNA
molecules in single file order
Transient chemical protection of DNA primer terminus (to
prevent elongation and excision) permits only a 20 min
window to sequence unmodified DNA
Hence we combine phi29 DNAP dependent
template with an improved blocking oligomer
strategy.
.
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Blocking oligomers allow the formation of phi29 DNAP-DNA
complexes that were enzymatically inactive in the presence
of dNTPs and Mg2+ for at least 5 hours.
25nt
70 nt
23nt3
5
3
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(i) the open channel;
(ii)nanopore capture of a polymerase-DNA complex with a blockingoligomer bound;
(iii) mechanical unzipping of the blocking oligomer promoted
by the applied voltage, which ratchets the DNA template forward
through the nanopore (this gives rise to the first 35-pA current peak as
the abasic insert traverses the major pore constriction); (iv) release of the blocking oligomer, which exposes the 3-OH terminus of the DNA
primer within the polymerase active site; (v) DNA replication by phi29
DNAP, which ratchets the template in the reverse direction through the
nanopore, giving rise to the second 35-pA current peak; (vi) stalling of
DNA replication when the abasic residues of the template strand reach
the catalytic site of phi29 DNAP
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This model makes three testable predictions. First,traversal of the first 35-pA peak resulting from voltage-driven unzipping of
the blocking oligomer is independent of phi29 DNAP catalytic capability.
So it should be observed in the absence of the Mg2+ ions .
Second, traversal of the second 35-pA ionic current peak requires DNAreplication, it should be dependent upon the presence of Mg2+ .
The second 35-pA peak was not observed.
The third is that progression into the proposed replication-dependent peak
should be influenced by the chemical identity of the DNA primer terminus.
Substitution of the 3-OH terminus with a 3-H terminus should delay appearanceof the second 35-pA current peak by causing a stall as the primer-template
junction is positioned in the polymerase active site.
This prediction also proved to be correct
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ESTIMATING DNA TEMPLATE REGISTRY ERRORS IN THE
NANOPORE DURING PHI29 DNAPCONTROLLED
TRANSLOCATION
Movement from 29pA in either direction is equivalent to one nt displacement
a.(i) Correct read. The current rises from 27 pA to 29 pA and resides there for atleast 3 ms before advancing to 34 pA.
a.(ii) Deletion. The ionic current advances directly from 27 pA to 34 pA and fails
to reside at 29 pA for at least 3 ms (arrow).
(iii) Insertion. The ionic current trace advances from27 pA to 29 pA. It resides at
29 pA for at least 3 ms but then slips back to 27 pA for at least 3ms (arrow).
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CONCLUSION
We conclude that DNA substrates pre-bound to phi29 DNAP
can be protected from enzymatic modification for many
hours using blocking oligomers
DNA molecules are enzymatically modified only at the
nanopore, it is possible to combine all components of thereplication reaction in the nanopore chamber at one time
and run a lengthy analysis of many DNA templates without
further user intervention
These DNA template registry errors (1024.5% combinedprobability for insertions and deletions at a given position)
must
be reduced for a commercial nanopore sequencing device