Automated Forward and Reverse Ratcheting of DNA In

8/10/2019 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

Documents

Automated Forward and Reverse Ratcheting of DNA In