Methods and devices for single-molecule whole genome analysis

• US 9,536,041 B2
• Filed: 02/12/2013
• Issued: 01/03/2017
• Est. Priority Date: 06/30/2008
• Status: Active Grant

• Current Assignee: BioNano Genomics, Inc.
• Original Assignee: BioNano Genomics, Inc.
• Inventors: Xiao, Ming, Cao, Han
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US13/765,353
• Publication Number:
  US 20130240357A1
• Time in Patent Office: 1,421 Days
• Field of Search: 435/6.1, 435/287.2, 536/23.1, 536/24.3, 977/704
• US Class Current: 1/1
• 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 a first single-molecule DNA, comprising:

labeling a plurality of sequence-specific locations on the first single-molecule DNA with a labeling technique that does not comprise labeling a cut or nicked strand of the first single-molecule DNA;

linearizing at least a portion of the labeled first single-molecule DNA in a nanochannel by physical-entropic confinement of the first single-molecule DNA; and

detecting a recognizable pattern of label positions on the linearized first single-molecule DNA or portion thereof in the nanochannel, wherein said detecting comprises simultaneously detecting genomic sequence information and epigenomic pattern information in the same field of view on the linearized first single-molecule DNA or portion thereof,

wherein said detecting comprises detecting at least one of a fluorescent signal, a chemoluminescent signal, an electromagnetic signal, an electrical signal, or a potential difference, and wherein detecting said genomic sequence information comprises detecting a signal, wavelength, or signal and wavelength different from detecting said epigenomic pattern information.

2. The method of claim 1, wherein the recognizable pattern of labels comprises a pattern of one or more selected from the group consisting of:

a structure, a sequence assembly, a genetic map, a cytogenetic map, a methylation pattern, a physiological characteristic, a location of a CpG island, an epigenomic pattern, and a combination thereof.

3. The method of claim 1, wherein the first single-molecule DNA is double stranded.

4. The method of claim 1, wherein the labeling is effected with one or more selected from the group consisting of 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, and any combination thereof.

5. The method of claim 1, wherein the labeling is effected with a methyltransferase.

6. The method of claim 1, wherein the label further comprises a fluorophore or quantum dot.

7. The method of claim 1, further comprising labeling at least one additional sequence-specific location on the first single-molecule DNA with a label that differs from the label of claim 1.

8. The method of claim 7, wherein one label identifies genomic information, and wherein the other label identifies epigenomic information.

9. The method of claim 1, wherein the nanochannel has a diameter of about 10 nm to about 200 nm.

10. The method of claim 1, wherein the labeling technique does not comprise hybridization of a sequence-specific nucleic acid probe.

11. A method of characterizing a first single-molecule DNA and a second single-molecule DNA, comprising:

labeling a plurality of sequence-specific locations on the first single-molecule DNA and the second single-molecule DNA with a labeling technique that does not comprise labeling a cut or nicked strand of either the first single-molecule DNA or the second single-molecule DNA;

linearizing at least a portion of the labeled first single-molecule DNA and a portion of the labeled second single-molecule DNA in a nanochannel, wherein said linearizing is by physical-entropic confinement of the first single-molecule DNA; and

detecting a recognizable pattern of label positions on the linearized first single-molecule DNA or portion thereof and on the linearized second single-molecule DNA or portion thereof, wherein said detecting comprises simultaneously detecting genomic sequence information and epigenomic pattern information in a first same field of view on the first single-molecule DNA and simultaneously detecting genomic sequence information and epigenomic pattern information in a second same field of view on the linearized second single-molecule DNA, and

wherein said detecting comprises detecting at least one of a fluorescent signal, a chemoluminescent signal, an electromagnetic signal, an electrical signal, or a potential difference, and wherein detecting said genomic sequence information comprises detecting a signal, wavelength, or signal and wavelength different from detecting said epigenomic pattern information.

12. The method of claim 11, wherein the first and second single-molecule DNAs are each double stranded.

13. The method of claim 12, wherein the labeling is effected with one or more selected from the group consisting of 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, and a combination thereof.

14. The method of claim 12, wherein the labeling is effected with a methyltransferase.

15. The method of claim 12, wherein the label further comprises a fluorophore or quantum dot.

16. The method of claim 12, further comprising labeling at least one additional sequence-specific location on the first single-molecule DNA with a label that differs from the label of claim 12.

17. The method of claim 16, wherein one label identifies genomic information, and wherein the other label identifies epigenomic information.

18. The method of claim 11, wherein the nanochannel has a diameter of about 10 nm to about 200 nm.

19. The method of claim 11, wherein the labeling technique does not comprise hybridization of a sequence-specific nucleic acid probe.

20. A method of characterizing a single-molecule DNA, comprising:

labeling at least a portion of the single-molecule DNA with a methyltransferase-mediated labeling technique that does not comprise labeling a cut or nicked strand of the DNA;

maintaining at least the portion of the labeled single-molecule DNA in a linearized form in a nanochannel; and

simultaneously detecting a plurality of signals in a same field of view from the linearized labeled single-molecule DNA or portion thereof, wherein said detecting comprises simultaneously detecting genomic sequence information and epigenomic pattern information in the same field of view on the labeled single-molecule DNA,

wherein said detecting comprises detecting at least one of a fluorescent signal, a chemoluminescent signal, an electromagnetic signal, an electrical signal, or a potential difference, and wherein detecting said genomic sequence information comprises detecting a signal, wavelength, or signal and wavelength different from detecting said epigenomic pattern information.

21. The method of claim 20, wherein the single-molecule DNA is double stranded.

22. The method of claim 21, further comprising:

measuring the intensity of the at least one signal, wherein the at least one signal comprises a signal of one or more selected from the group consisting of;

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, and a combination thereof.

23. The method of claim 21, further comprising:

detecting a pattern of labels on the labeled double-stranded DNA, wherein the pattern of labels comprises a pattern of one or more selected from the group consisting of;

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, and a combination thereof.