Characterizing stretched polynucleotides in a synthetic nanopassage

1. A method of characterizing at least a portion of a double-stranded polynucleotide, said method comprising:

providing a membrane having a nanopassage that defines a confine region, wherein said membrane separates a first fluid compartment from a second fluid compartment, and said nanopassage is in fluid communication with the first and second compartments;

providing said double-stranded polynucleotide to said first fluid compartment;

establishing a driving voltage bias that is greater than a threshold voltage across said membrane to force a portion of said double-stranded polynucleotide into said nanopassage, wherein said portion of said double-stranded polynucleotide has a confined portion positioned within said confine region and said confine region deforms said double-stranded polynucleotide by increasing axial rise from an undeformed axial rise value to a deformed axial rise value in said confine region or a region adjacent thereto;

monitoring an electrical current through said nanopassage;

identifying a confine state from said a monitored electrical current from said monitoring step, wherein said confine state corresponds to said confined portion containing a confined nucleotide base-pair of said polynucleotide in said confine region;

reducing said driving voltage bias to a holding voltage that is less than or equal to said threshold voltage, thereby trapping said confined nucleotide base-pair in said nanopassage confine region for a trapping time, wherein said trapping time is selected from a range that is greater than or equal to 10 ns and less than or equal to 1 second; and

measuring a nucleotide base-pair dependent current blockade through said nanopassage confine region having said confined nucleotide base-pair during said trapping time, to characterize the confined nucleotide base-pair, thereby characterizing at least a portion of said polynucleotide.

2. The method of claim 1 , further comprising:

establishing a translocation voltage bias that is greater than the threshold voltage to force said confined nucleotide base-pair out of said confine region in a direction that is toward said second compartment;

repeating said monitoring, identifying, reducing and measuring steps to thereby characterize a confined nucleotide base-pair that is at a position upstream from the previously characterized confined nucleotide.

3. The method of claim 2 , wherein the characterized confined nucleotides base-pair are nucleotide base-pairs adjacent to each other in said polynucleotide.

4. The method of claim 2 , further comprising repeating the steps of claim 2 to characterize a contiguous portion of said polynucleotide, wherein said contiguous portion corresponds to at least 10% of the entire length of said polynucleotide.

5. The method of claim 4 , wherein said contiguous portion corresponds to the entire length of said polynucleotide.

6. The method of claim 2 , wherein said translocation voltage is less than said driving voltage bias.

7. The method of claim 2 , wherein said holding voltage is applied for a holding time sufficient to provide high-fidelity assessment of said confined nucleotide base-pair.

8. The method of claim 2 wherein said polynucleotide travels in a direction from said first compartment to said second compartment at a translocation velocity that is greater than or equal to 1 nucleotide per 10 nanoseconds or greater than or equal to 1 nucleotide base pair per 10 nanoseconds.

9. The method of claim 2 , further comprising: diagnosing a medical condition for a patient from which the polynucleotide is obtained.

10. The method of claim 9 wherein the medical condition relates to a specific polynucleotide sequence.

11. The method of claim 9 , wherein the medical condition relates to a methylation parameter.

12. The method of claim 2 , wherein the translocation voltage bias is a voltage pulse having a duration that is less than or equal to 1 μs.

13. The method of claim 2 , wherein the magnitude of said translocation voltage bias is at least two times greater than said holding voltage.

14. The method of claim 1 , wherein said polynucleotide translocates unidirectionally from said first compartment to said second compartment.

15. The method of claim 1 , further comprising determining said threshold voltage for said nanopassage and said polynucleotide.

16. The method of claim 1 , wherein said characterizing is one or more of identifying a nucleotide-type, a nucleotide base-pair type or nucleotide methylation state.

17. The method of claim 1 wherein said characterization is methylation content, methylation pattern, or methylation content and pattern.

18. The method of claim 1 , wherein said characterization is determining at least a portion of said polynucleotide sequence.

19. The method of claim 1 wherein said deformed axial rise value is at least 20% greater than the undeformed axial rise value.

20. The method of claim 1 , wherein said deformed axial rise value is selected from a range that is greater than or equal to 0.34 nm and less than or equal to 0.7 nm.

21. The method of claim 1 , wherein said characterization comprises determining the sequence of at least 2000 contiguous bases.

22. The method of claim 1 , wherein said confine region has a minimum cross-sectional area that is less than or equal to 4.4 nm 2 .

23. The method of claim 22 , wherein said nanopassage is a tapered nanopore having a maximum diameter that is less than or equal to 2.6 nm and a minimum diameter centered in said confine region that is selected from a range that is greater than or equal to 1 nm and less than or equal to 2.4 nm.

24. The method of claim 1 , wherein said membrane is a Si 3 N 4 membrane having a thickness selected from a range that is greater than or equal to 5 nm and less than or equal to 30 nm.

25. The method of claim 1 , wherein one nucleotide of said confined nucleotide base-pair is uniquely identified with one strand of said double stranded polynucleotide.

26. The method of claim 1 , further comprising the step of obtaining a high fidelity measure of the nucleotide base-pair dependent current blockade by measuring the nucleotide base-pair dependent current blockade over the trapping time and calculating an average nucleotide base-pair dependent current blockade over the trapping time.

27. A method of trapping a portion of a double-stranded polynucleotide in a membrane nanopassage, said method comprising the steps of:

providing a membrane having a nanopassage that defines a confine region, wherein said membrane separates a first fluid compartment from a second fluid compartment, and said nanopassage is in fluid communication with the first and second compartments;

providing said double-stranded polynucleotide to said first fluid compartment;

determining a threshold voltage for said membrane and said double-stranded polynucleotide;

establishing a driving voltage bias across said membrane that is greater than said threshold voltage, to force a portion of said double-stranded polynucleotide into said nanopassage confine region, wherein said portion of said double-stranded nucleotide in said confine region is deformed and said confine region deforms said double-stranded polynucleotide by increasing axial rise from an undeformed axial rise value to a deformed axial rise value in said confine region or a region adjacent thereto; and

decreasing said driving voltage bias to a holding voltage bias, wherein said holding voltage bias is less than said threshold voltage, thereby trapping said polynucleotide portion in said nanopassage confine region for a trapping time, wherein said trapping time is selected from a range that is greater than or equal to 10 ns and less than or equal to 1 second, wherein at least one nucleotide base-pair is fixably positioned in said nanopassage confine volume during said trapping time.

28. The method of claim 27 , further comprising:

measuring a blockade current through said nanopassage having at least one nucleotide base-pair positioned in said confine volume;

sequentially forcing said polynucleotide through said confine volume nucleotide base-pair by nucleotide base-pair by switching an electric field from a translocation voltage bias that is greater than said threshold voltage to said holding voltage bias at a switching frequency,

wherein said holding voltage bias is applied when a nucleotide base-pair is positioned in said confine region, and said sequentially forcing step provides a nucleotide base-pair stepwise movement of said polynucleotide through said confine region in a direction from said first compartment to said second compartment so that every nucleotide base-pair within a contiguous length of said polynucleotide is trapped in said confine region and said blockade current is measured for each trapped nucleotide base-pair.

29. The method of claim 28 , wherein the holding voltage bias corresponds to no voltage difference across said membrane.

30. The method of claim 27 , wherein said holding voltage is applied for a holding time that is sufficient to provide high-fidelity measurement of said blockade current for said nucleotide base-pair positioned in said confine volume.

31. The method of claim 27 , wherein said nanopassage confine region that traps said portion of polynucleotide has a minimum cross-sectional area that is 2.56 nm 2 .

32. The method of claim 27 , wherein said polynucleotide is DNA having a length that is greater than or equal to 200 base pairs.

33. The method of claim 27 , wherein six or fewer base pairs are trapped in said nanopassage interior volume.

34. The method of claim 27 , wherein the nanopassage is a pore having a minimum diameter that is smaller than an average diameter of said polynucleotide that is trapped.

35. The method of claim 27 , further comprising measuring an electrical blockade current across said nanopassage for said trapped portion.

36. A method of sequencing a double-stranded polynucleotide, said method comprising:

providing a membrane having a nanopassage that defines a confine region having a minimum dimension that is less than an average axial diameter of said double-stranded polynucleotide, wherein said membrane separates a first fluid compartment from a second fluid compartment, and said nanopassage is in fluid communication with the first and second compartments;

providing said double-stranded polynucleotide to said first fluid compartment;

establishing a driving voltage bias that is greater than a threshold voltage across said membrane to force a portion of said double-stranded polynucleotide into said nanopassage, wherein said portion of said double-stranded polynucleotide has a confined portion positioned within said confine region;

monitoring an electrical current through said nanopassage;

identifying said confined portion as a confined nucleotide base-pair from a monitored electrical current from said monitoring step;

reducing said driving voltage bias to a holding voltage that is less than or equal to said threshold voltage, thereby trapping said confined nucleotide base-pair in said nanopassage confine region for a trapping time, wherein said trapping time is selected from a range that is greater than or equal to 10 ns and less than or equal to 1 second;

measuring a nucleotide-dependent current blockade through said nanopassage confine region having said confined nucleotide base-pair during said trapping time, to identify the confined nucleotide base-pair, thereby characterizing at least a portion of said polynucleotide;

establishing a translocation voltage bias that is greater than the threshold voltage to translocate said polynucleotide in a direction that is toward said second compartment, wherein said translocation moves said polynucleotide by one base-pair through the confine region;

repeating said monitoring, identifying, reducing and measuring steps to thereby identify a confined nucleotide base-pair that is at a single base-pair sequential position difference from the previously characterized confined nucleotide base-pair, thereby sequencing at least a portion of said double-stranded polynucleotide.

37. The method of claim 36 wherein the measuring step is repeated over the entire polynucleotide length, thereby sequencing the entire polynucleotide.