Random array dna analysis by hybridization

• US 20070037152A1
• Filed: 02/26/2004
• Published: 02/15/2007
• Est. Priority Date: 02/26/2003
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Inventors: Drmanac, Radoje
• Granted Patent: US 8,105,771 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00479 Means for mixing reactants or products in the reaction vessels
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 20/00 Nanooptics, e.g. quantum optics or photonic crystals
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/619 being a video camera
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• 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 2201/06113 Coherent sources; lasers
• CPC Class Codes: G01N 2223/413 sensor array [CCD]

Abstract

The invention relates to methods and devices for analyzing single molecules, i.e. nucleic acids. Such single molecules may be derived from natural samples, such as cells, tissues, soil, air and water without separating or enriching individual components. In certain aspects of the invention, the methods and devices are useful in performing nucleic acid sequence analysis by probe hybridization.

Claims

1. A method to analyze nucleic acids comprising:

(a) hybridizing a population of oligonucleotide probes of the same informative region sequence to an array of single molecule target nucleic acid under conditions that produce on average, more efficient hybridization to full-match than to mismatch sequences;

(b) collecting a signal produced by multiple consecutive hybridization of oligonucleotides molecules to each target molecule; and

(c) analyzing said signal.

2. The method of claim 1 further comprising:

(a) ligating at least two oligonucleotide probes that are hybridized to the target nucleic acid molecule.

6. The method of claim 1, wherein the probes or sets of probes are consecutively hybridized to the array.

7. The method of claim 1, wherein the probes are selected from the group consisting of an incomplete set of probes, a complete set of probes, pools of probes, and informative probe pools.

8. The method of claim 1, wherein the probes are comprised of at least one modified or universal base.

9. The method of claim 1, wherein the target nucleic acid is detectably labeled with a label selected from the group consisting of:

a fluorophore, a nanotag, a chemiluminescent label, a quantum dot, a quantum bead, a fluorescent protein, a dendrimer with a fluorescent label, a micro-transponder, an electron donor molecule or molecular structure, an electron acceptor molecule or molecular structure, and a light-reflecting particle.

10. The method of claim 1, wherein at least one probe is detectably labeled with a label selected from the group consisting of:

a fluorophore, a nanotag, a chemiluminescent label, a quantum dot, a quantum bead, a fluorescent protein, a dendrimer with a fluorescent label, a micro-transponder, an electron donor molecule or molecular structure, an electron acceptor molecule or molecular structure, and a light-reflecting particle.

11. The method of claim 1, wherein a mega-pixel CCD camera is used to collect said signal.

12. The method of claim 1, wherein the single molecule target nucleic acids are randomly arrayed on an optically clear substrate at a density of 0.1-10, uM.

13. The method of claim 12, wherein the substrate is a glass slide.

14. The method of claim 1, wherein the single molecule target nucleic acids are randomly arrayed on an optically clear substrate at a density of at least one molecule per pixel.

15. The method of claim 1, wherein the target nucleic acids are obtained from a pathogen.

16. The method of claim 1, wherein the randomly arrayed target nucleic acids molecules are in situ amplified.

17. The method of claim 1, wherein the analysis includes counting occurrences of targets with the same or shared sequence in the array.

3. A method to analyze nucleic acids comprising:

(a) hybridizing one or more probes from a first set of detectably labeled oligonucleotide probes to a random array of target nucleic acids;

(b) hybridizing one or more probes from a second set of detectably labeled or unlabeled oligonucleotide probes to said target nucleic acids;

(c) ligating at least one probe from each said set that are hybridized to said target nucleic acid; and

(d) detecting and analyzing said ligated probe(s).

4. A method to analyze nucleic acids comprising:

(a) hybridizing a population of a first set of detectably labeled oligonucleotide probes to an array of target nucleic acids;

(b) hybridizing a population of a second set of detectably labeled oligonucleotide probes to said arrayed target nucleic acids;

(c) ligating at least two detectably labeled probes that are hybridized to said target nucleic acid molecule; and

(d) detecting a fluorescence resonance energy transfer (FRET) signal between said labeled probes.

5. The method of claim 4, further comprising:

(a) collecting multiple FRET signals; and

(b) comparing the collection to a pattern indicative of a pathogen.

18. A method of amplifying a nucleic acid comprising the steps of:

(a) binding of an invading oligonucleotide to one end of the target DNA;

(b) hybridizing of a first primer oligonucleotide to a first available single stranded site of the target DNA;

(c) hybridizing of a second primer oligonucleotide to a second or opposite single stranded site of the target DNA; and

(d) repeating steps 1-3.

19. A kit comprising, a first set of probes associated with a FRET acceptor molecule, a second set of probes optionally associated with a FRET donor molecule, a ligating reagent, and substrates for forming random target DNA arrays.

20. A kit comprising, a first set of probes associated with a FRET acceptor molecule, a second set of probes associated with a FRET donor molecule, and a ligating reagent.

21. A composition or mixture comprising a first set of probes associated with a FRET acceptor molecule, a second set of probes associated with a FRET donor molecule, and a ligating molecule.

22. An apparatus comprising:

(a) a plurality of reservoirs for probe solution; and

(b) a reaction chamber for hybridizing and displaying a random array of target DNA.

23. An apparatus comprising:

(a) a reservoir or an inlet port for a sample;

(b) one or more reservoirs for chemical reagents or solutions for isolating and/or fragmenting DNA from the sample;

(c) a plurality of reservoirs for probe solutions;

(d) optionally a mixing chamber or tube in which reagents are mixed with the sample and/or the probes; and

(e) a reaction chamber for displaying a random array of target DNA from said sample.