Methods and devices for single-molecule whole genome analysis

• US 8,628,919 B2
• Filed: 06/30/2009
• Issued: 01/14/2014
• Est. Priority Date: 06/30/2008
• Status: Active Grant

• Current Assignee: BioNano Genomics, Inc.
• Original Assignee: BioNano Genomics, Inc.
• Inventors: Xiao, Ming, Deshpande, Parikshit A., Cao, Han, Austin, Michael, Vijayan, Kandaswamy, Sharonov, Alexey Y., Boyce-Jacino, Michael
• Primary Examiner(s): Nguyen, Dave
• Assistant Examiner(s): BHAT, NARAYAN KAMESHWAR
• Application Number:
  US13/001,697
• Publication Number:
  US 20110171634A1
• Time in Patent Office: 1,659 Days
• Field of Search: 435/6.1, 435/6.11, 435/287.2, 536/23.1, 977/704
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2537/1376 Displacement by an enzyme
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 Detection of binding sites or motifs

Abstract

Provided are methods and devices for single-molecule genomic analysis. In one embodiment, the methods entail processing a double-stranded nucleic acid and characterizing said nucleic acid. These methods are useful in, e.g., determining structural variations and copy number variations between individuals.

Claims

1. A method of characterizing DNA, comprising:

processing a double-stranded DNA comprising a first DNA strand and a second DNA strand to give rise to an unhybridized flap of the first DNA strand and a corresponding region on the second DNA strand,

the unhybridized flap comprising about 10 to about 1000 bases;

extending the first DNA strand along the corresponding region of the second DNA strand; and

labeling at least a portion of the unhybridized flap, a portion of the extended first DNA strand, or both.

2. The method of claim 1, wherein the processing comprises nicking the first DNA strand.

3. The method of claim 2, wherein the nicking is effected at one or more sequence specific locations.

4. The method of claim 2, wherein the nicking is effected at one or more non-specific locations.

5. The method of claim 1, wherein the labeling is accomplished by (a) binding at least one complementary probe to at least a portion of the unhybridized flap, the probe comprising one or more tags, (b) extending the first DNA strand along the corresponding region of the second DNA strand with one or more nucleotides comprising one or more tags, or any combination of (a) and (b).

6. The method of claim 5, further comprising obtaining sequence information derived from one or more tags.

7. The method of claim 5, wherein labeling comprises attaching a tag comprising a fluorophore, a quantum dot, a dendrimer, a nanowire, a bead, a peptide, a protein, a magnetic bead, a methyl group, a methyltransferase, a non-cutting restriction enzyme, a zinc-finger protein, an antibody, a transcription factor, a DNA binding protein, a hairpin polyamide, a triplex-forming oligodeoxynucleotide, a peptide nucleic acid, or any combination thereof.

8. The method of claim 7, further comprising at least partially linearizing at least a portion of the double-stranded DNA comprising an unhybridized flap, a portion of the extended region of the first DNA strand, or both.

9. The method of claim 8, further comprising measuring the distance between two or more tags.

10. The method of claim 9, further comprising correlating the distance to a structure, a sequence assembly, a genetic or cytogenetic map, a methylation pattern, a location of a cpG island, an epigenomic pattern, a physiological characteristic, or any combination thereof.

11. The method of claim 8, wherein the linearization is effected by confinement of the DNA in a channel.

12. The method of claim 7, further comprising measuring the intensity of at least one signal from at least one tag.

13. The method of claim 12, further comprising correlating the intensity of the at least one signal to a structure, a sequence assembly, a genetic or cytogenetic map, a methylation pattern, a physiological characteristic, a location of a cpG island, an epigenomic pattern, or any combination thereof.

14. The method of claim 5, further comprising detecting one or more signals from one or more tags.

15. The method of claim 1, wherein the flap comprises at least a portion of a base sequence of interest on the double-stranded DNA.

16. The method of claim 1, wherein the unhybridized flap of the first DNA strand, the corresponding region of the second DNA strand, or both, comprises at least a portion of the double-stranded DNA that flanks a base sequence of interest on the double-stranded DNA.

17. The method of claim 1, further comprising obtaining epigenomic information from the DNA.

18. A method of characterizing DNAs, comprising:

labeling, on a first double-stranded DNA, two or more sequence-specific locations on the first DNA;

labeling, on a second double-stranded DNA, the two or more corresponding sequence-specific locations on the second DNA;

linearizing at least a portion of the first double-stranded DNA;

linearizing at least a portion of the second double-stranded DNA; and

comparing the distance between two or more labels on the first, linearized double-stranded DNA to the distance between the corresponding labels on the second, linearized double-stranded DNA, wherein the labeling is accomplished by nicking a first strand of a double-stranded DNA so as to give rise to (a) an unhybridized flap of the first strand and (b) a corresponding region in the second strand of the double-stranded DNA, wherein the unhybridized flap comprises about 10 to about 1000 bases.

19. The method of claim 18, further comprising exposing the flap to a labeled probe complementary to at least a portion of the probe, extending the first strand of the DNA along the corresponding region of the second strand with one or more labeled bases, or both.

20. The method of claim 18, wherein the labeling is accomplished by exposing the first and second double-stranded DNAs to a non-cutting restriction enzyme, a methyltransferase, a zinc finger protein, an antibody, a transcription factor, a DNA binding protein, a hairpin polyamide, a triplex-forming oligodeoxynucleotide, a peptide nucleic acid, or any combination thereof.

21. The method of claim 20, wherein the non-cutting restriction enzyme comprises a tag.

22. The method of claim 18, further comprising correlating the distance to a structure, a sequence assembly, a genetic or cytogenetic map, a methylation pattern, a physiological characteristic, a location of a cpG island, an epigenomic pattern, or any combination thereof, of the DNA.

23. The method of claim 18, further comprising obtaining epigenomic information from a DNA.

24. A method of characterizing DNA, comprising:

labeling, on a first double-stranded DNA, one or more sequence-specific locations on the first double-stranded DNA;

labeling, on a second double-stranded DNA, the corresponding one or more sequence-specific locations on the second double-stranded DNA;

linearizing at least a portion of the first double-stranded DNA;

linearizing at least a portion of the second double-stranded DNA; and

comparing the intensity of a signal of a label of the first, linearized double-stranded DNA to the intensity of the signal of a label of the second, linearized double-stranded DNA, wherein the labeling is accomplished by nicking a first strand of each of the first and second double-stranded DNAs so as to give rise to (a) an unhybridized flap of the first strand and (b) a corresponding region in the second strand of the corresponding double-stranded DNA, wherein the unhybridized flap comprises about 10 to about 1000 bases.

25. The method of claim 24, further comprising exposing the flap to a labeled probe complementary to at least a portion of the probe, extending the first strand of the DNA along the corresponding region of the second DNA strand with one or more labeled bases, or both.

26. The method of claim 24, further comprising correlating the signal intensities to a structure, a sequence assembly, a genetic or cytogenetic map, a methylation pattern, a physiological characteristic, a location of a cpG island, an epigenomic pattern, or any combination thereof, of the DNA.

27. The method of claim 24, further comprising obtaining epigenomic information from the DNA.

28. A method of characterizing a macromolecule, comprising:

translocating a macromolecule comprising at least one flap extending therefrom along a channel having at least one constriction disposed therein, wherein the flap comprises about 10 to about 1000 bases; and

detecting at least one signal corresponding to the passage of the at least one flap of the macromolecule through the at least one constriction of the channel.

29. The method of claim 28, wherein the flap comprises a label.

30. The method of claim 28, wherein the macromolecule comprises a label.

31. The method of claim 28, wherein the macromolecule comprises double-stranded DNA or chromatin fibers.

32. The method of claim 28, further comprising translocating the at least one flap through the at least one constriction more than once.

33. A method of characterizing a macromolecule, comprising:

labeling at least a portion of a macromolecule, wherein the labeled macromolecule comprises at least a flap comprising about 10 to about 1000 bases;

immobilizing the macromolecule;

disposing at least a portion of the macromolecule within a channel such that at least a portion of the macromolecule is linearized within the channel; and

detecting at least one signal related to the labeled portion of the macromolecule.

34. The method of claim 33, wherein the macromolecule is bound to at least one bead, the molecule being immobilized by the at least one bead being caught by a constriction smaller in cross-section than the bead.

35. The method of claim 33, wherein the immobilization is accomplished by chemically tethering the macromolecule to a surface, by magnetically immobilizing the macromolecule, by optically trapping the macromolecule, or any combination thereof.

36. The method of claim 33, wherein the macromolecule comprises at least one dielectric modification and is immobilized by an optical trap.

37. The method of claim 33, wherein the channel is sized so as to effect linearization of the macromolecule.

38. A method of characterizing a nucleic acid polymer, comprising:

labeling one or more regions of a nucleic acid polymer with one or more sequence-specific motif labels, wherein the labeled nucleic acid polymer comprises at least a flap comprising about 10 to about 1000 bases;

correlating one or more signals from one or more of the sequence-specific motif labels to the position of the one or more sequence-specific motif labels of the nucleic acid polymer;

sequencing one or more segments of the nucleic acid polymer, the one or more segments including one or more of the sequence specific motif labels of the nucleic acid polymer; and

comparing one or more signals of one or more sequenced segments to one or more corresponding signals of the labeled nucleic acid polymer so as to develop relative locations within the nucleic acid polymer, of two of more sequenced segments.

39. The method of claim 38, wherein the labeling is accomplished by forming a flap in the nucleic acid polymer and labeling the flap, the region vacated by the flap, or any combination thereof.

40. The method of claim 38, wherein the correlating comprises linearizing at least one labeled portion of the nucleic acid polymer.

41. The method of claim 40, wherein the linearizing comprises disposing the at least one labeled portion of the nucleic acid polymer in a channel capable of linearizing the at least one labeled portion of the nucleic acid polymer.

42. The method of claim 38, further comprising application of a gradient to the at least one labeled portion of the nucleic acid polymer so as to linearize the at least one labeled portion of the nucleic acid polymer, to maintain the at least one labeled portion of the nucleic acid polymer in linear form, or any combination thereof.

43. The method of claim 38, wherein the correlating comprises determining the distance between two or more labels, comparing the intensity of signals evolved from two or more labels, or any combination thereof.

44. The method of claim 38, wherein the sequencing comprises Sanger sequencing, Maxam-Gilbert sequencing, dye terminator sequencing, in vitro clonal amplification, sequencing by synthesis, sequencing by hybridization, sequencing by ligation or any combination thereof.

45. The method of claim 38, wherein a segment comprises from about 5 to about 30 kilobases.

46. The method of claim 38, wherein the comparing comprises aligning one or more labeled, sequenced segments against the labeled nucleic acid polymer such that a sequence-specific motif label of the labeled, sequenced segment is in register with the corresponding sequence-specific motif label of the labeled nucleic acid polymer.