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AUTOMATED FORWARD AND

REVERSE RATCHETING OF DNA IN


A NANOPORE AT 5-A PRECISION
Gerald M Cherf et al.

GROUP MEMBERS
Pranav Varma
Nihal Sangeeth
Ghanim Fajish
Alex Johny

INTRODUCTION TO NANOPORE SEQUENCING


The motive is to find the order in which
nucleotides 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

COMMON NANOPORE : ALPHA HAEMOLYSIN

Alpha haemolysin (HL), from bacteria that


causes lysis of red blood cells, has been
studied 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 ~10 nm 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.

6 FEATURES
Automated capture and processing of genomic DNA
templates in single file order from a heterogeneous
mixture 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 homopolymeric
regions

APPARATUS
Pore forming protein -haemolysin ( -HL)
-HL nanopore is inserted in a lipid bilayer
that separates two wells each filled with 100L
KCl buffer.
Negatively charged ssDNA added to cis well
A voltage is applied between the two wells

ENZYME MOTOR COUPLING


Means to slow down template strand
movement(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

DNAP AS ENZYME MOTOR COUPLES


T7 DNAP binds to ssDNA;catalyses nt
additions that advances template strand
through the pore(applied voltage-80mV)
Use of T7 DNAP in sequencing not practical
for 2 reasons

i.

ii.

At most 3 sequential ionic current steps observed


before t7 DNAP dissociation from template
To remove blocking oligomer and bind t7 DNAP ,the
DNA was tethered in pore and driven back and forth
by reversing polarity at 10 ms intervals resulting in
high crosstalk between pores

DNAP AS ENZYME MOTOR COUPLES


Experimental data suggest phi29(a B-family
polymerase) bounds to DNA ~10000 times longer
than A-family polymerases(T7 DNAP)
Controlled sequential movement of atleast 50
base through nanopore from a precise starting
point in a primer strand without active voltage
control
Rate of elongation and template displacement
was tens of milliseconds/nt

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.
.

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.
3

25nt
70 nt

23nt

5
3

(i) the open channel;


(ii)nanopore capture of a polymerase-DNA complex with a blocking
oligomer 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

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 DNA
replication, 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 appearance
of 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

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 at
least 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).

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 the
replication 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% combined
probability for insertions and deletions at a given position)
must
be reduced for a commercial nanopore sequencing device