Methods of macromolecular analysis using nanochannel arrays

• US 9,310,376 B2
• Filed: 03/03/2014
• Issued: 04/12/2016
• Est. Priority Date: 03/28/2007
• Status: Active Grant

• Current Assignee: BioNano Genomics, Inc.
• Original Assignee: BioNano Genomics, Inc.
• Inventors: Cao, Han, Deshpande, Parikshit A, Boyce-Jacino, Michael, Austin, Michael D
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US14/195,474
• Publication Number:
  US 20140249039A1
• Time in Patent Office: 771 Days
• Field of Search: 435/6.1, 435/6.11, 435/287.2, 536/23.1, 536/24.3, 977/704
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0663 Stretching or orienting elongated molecules or particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/041 Connecting closures to device or container
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/0896 Nanoscaled
• CPC Class Codes: B01L 2300/161 Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2563/107 : fluorescence
• CPC Class Codes: C12Q 2600/172 : Haplotypes
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 : Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 33/582 : with fluorescent label

Abstract

Methods of analyzing features such as the physical size of macromolecules or biomarkers along large genomic DNA molecules were disclosed as wen as the devices for carrying out such high throughput analysis in a massively parallel fashion. Methods of fabricating such devices are also disclosed.

Claims

1. A method of detecting a copy number abnormality in a genome of interest, the method comprising:

obtaining a first sample comprising one or more isolated nucleic acid molecules of the genome of interest;

labeling one or more regions, including at least a first region, of the isolated nucleic acid molecules of the genome of interest;

translocating at least a portion of at least the first region of each of the labeled isolated nucleic acid molecules through a fluidic nanochannel segment disposed substantially parallel to a substrate surface, wherein the fluidic nanochannel segment is capable of containing and elongating at least a portion of the region of the nucleic acid, and wherein the fluidic nanochannel segment has a characteristic cross-sectional dimension of less than about 1000 nm and a length of at least about 10 nm, wherein said translocating comprises introducing the labeled isolated nucleic acid molecules into separate fluidic nannochannel segments each having a cross-sectional diameter of less than about 200 nm so as to at least substantially linearize at least a part of the labeled nucleic acid molecules;

during said translocating, capturing through a viewing window a total length of time that signals corresponding to each first region of the labeled individual isolated nucleic acid molecules of the genome of interest are detected through the viewing window, said total length of time correlating to the speed at which said labeled isolated nucleic acid molecules move through the fluidic nanochannel segment, thereby ascertaining a length of nucleic acids corresponding to the first region; and

comparing the total length of time of signals of each first region of the labeled individual isolated nucleic acid molecules of the genome of interest to a total length of time of signals of a control, whereby a copy number of at least the first region of the isolated nucleic acid molecules of the genome of interest is ascertained from the comparing of lengths of time.

2. The method of claim 1, wherein the control comprises a second region of the genome of interest.

3. The method of claim 2, further comprising ascertaining a copy number of the second region of the control, and then comparing the copy numbers of the first region and the second region.

4. The method of claim 3, wherein the first region is a trisomy-linked region and the second region of the control is different from the trisomy-linked region.

5. The method of claim 4, wherein labeling comprises binding a first label to a trisomy-linked chromosome comprising the trisomy-linked region of the first region and binding a second label to the second region of the control, wherein the second region of the control is on a chromosome other than the trisomy-linked chromosome.

6. The method of claim 5, wherein comparing the total length of time of signals of each first region of the labeled isolated nucleic acid molecules of the genome of interest to the total length of time of signals of the control comprises calculating a ratio of the total length of time of signals from each first region to the total length of time of signals from the second region of the control, wherein the ratio varies with copy number of the respective first and second regions.

7. The method of claim 6, wherein a ratio of about 1.5:

1 is indicative of trisomy in the sample of interest.

8. The method of claim 1, wherein the copy number abnormality comprises a trisomy.

9. The method of claim 1, wherein labeling one or more regions, including at least the first region, of the isolated nucleic acid molecules of the genome of interest comprises:

contacting the isolated nucleic acid molecules of the genome of interest with;

a first labeled probe of known length L1, wherein the first labeled probe is complementary to a genomic sequence of the control, wherein the copy number of the control is known; and

a second labeled probe of known length L2, wherein the second labeled probe is specific to the first region;

detecting binding between the first labeled probe and the genomic sequence of the control and between the second labeled probe and the first region; and

ascertaining the total length of the hybridization signals that correspond to the first labeled probe (S1) and the second labeled probe (S2),

wherein the copy number of at least the first region is calculated by calculating the ratios N1=S1/L1 and N2=S2/L2, wherein N1 corresponds to the copy number of the genomic sequence of the control and N2 corresponds to the copy number of the first region.

10. The method of claim 1, wherein capturing a total length of time that signals corresponding to each first region are detected through the viewing window further comprises identifying a haplotype of the genome of interest.

11. The method of claim 1, wherein the total length of time of signals of the control comprises information from a different genome than the genome of interest.

12. The method of claim 1, wherein labeling one or more regions, including the first region, of the one or more isolated nucleic acid molecules of the genome of interest comprises contacting the isolated nucleic acid molecules with a first label of a known length, wherein the first label is specific to the first region, and;

and wherein ascertaining the copy number of at least the first region of the isolated nucleic acid molecules comprises determining a first ratio of the total length of time of signals of each first region to the known length of the first label, so that the first ratio corresponds to a copy number of at least the first region of the isolated nucleic acid molecules.

13. The method of claim 12, further comprising comparing the first ratio to a second ratio, wherein the second ratio corresponds to a copy number of a sequence of the control having a known length and a known copy number, and wherein a difference between the first ratio and the second ratio indicates a copy number abnormality of nucleic acids corresponding to the first region.

14. The method of claim 13, further comprising:

contacting the isolated nucleic acid molecules with a second label of known length, wherein the second label is specific to the sequence of the control.

15. The method of claim 13, wherein the second ratio comprises labeling pattern information from a different genome than the genome of interest.

16. The method of claim 13, wherein the sequence of the control is the same as the sequence of the first region, and the second ratio comprises labeling pattern information from a different genome than the genome of interest.

17. The method of claim 12, wherein the first region comprises a repeating sequence.

18. The method of claim 1, wherein the first region comprises separate portions whose length per normal genome is known.

19. The method of claim 1, wherein the control comprises separate portions whose total length per normal genome is known.

20. The method of claim 1, wherein labeling comprises labeling with at least one of a fluorescent label, a magnetic label, or a radioactive label.

21. The method of claim 1, wherein the labeled isolated nucleic acid molecules are from a single cell.

22. The method of claim 1, further comprising ascertaining, based on total length of time of the signals, a copy number of at least the first region on a single isolated nucleic acid molecule of the genome of interest.

23. The method of claim 1, wherein the control comprises a nucleotide sequence from a different genome than the genome of interest.

24. The method of claim 1, wherein the viewing window comprises a slit, a porthole, a square, or any combination thereof.

25. The method of claim 1, wherein the viewing window is capable of permitting optical inspection of at least a portion of the contents of the fluidic nanochannel segment.

26. The method of claim 1, wherein the viewing window is capable of permitting optical inspection of all of the contents of the fluidic nanochannel segment.

27. The method of claim 1, wherein the viewing window is removable.