Random array DNA analysis by hybridization

• US 20090036316A1
• Filed: 10/31/2007
• Published: 02/05/2009
• Est. Priority Date: 02/26/2003
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Radoje T.
• Granted Patent: US 7,906,285 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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

1-23. -23. (canceled)

24. A method to analyze nucleic acids comprising:

(a) hybridizing a population of oligonucleotide probes comprising the same region B_y to an array of target nucleic acids under reaction conditions optimized for multiple consecutive hybridization to full-match sequences;

(b) collecting a signal produced by multiple consecutive hybridization of oligonucleotide probes to the target nucleic acids on the array; and

(c) analyzing said signal.

25. The method of claim 24, wherein the region B_y is between 4 and 100 nucleotides in length.

26. The method of claim 24, wherein the oligonucleotide probes comprise at least one universal base at a terminal position.

27. A method to analyze target nucleic acids comprising:

(a) hybridizing a population of oligonucleotide probes comprising the same region B_y to an array of target nucleic acids under reaction conditions optimized for multiple consecutive hybridization to full-match sequences;

(b) ligating at least two oligonucleotide probes that are hybridized to the target nucleic acid molecules on the array;

(c) collecting a signal produced by the multiple consecutive hybridization of oligonucleotides molecules and probe ligation; and

(d) analyzing said signal.

28. The method of claim 27, wherein the region B_y is between 4 and 100 nucleotides in length.

29. The method of claim 28, wherein one or more of the probes is comprised of at least one universal base at a terminal position.

30. A method of determining the sequence of a target nucleic acid, comprising the steps of:

(a) hybridizing a first set of detectably-labeled oligonucleotide probes to a random array of nucleic acids generated from the target nucleic acid under reaction conditions optimized for multiple consecutive hybridization to full-match sequences;

(b) hybridizing a second set of detectably-labeled oligonucleotide probes to said target nucleic acids on the array under reaction conditions optimized for multiple consecutive hybridization to full-match sequences;

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

(d) detecting and analyzing said ligated probes;

wherein a sequence of the target molecule is assembled by ordering overlapping probe sequences that hybridize to the target molecule.

31. The method of claim 30, wherein a sequence of the target nucleic acid is assembled by ordering overlapping probe sequences and determining the probability of the assembled sequence from the hybridization efficiency of the incorporated probes.

32. The method of claim 30, wherein each probe within a set comprises the same region B_y, and wherein the probes comprising the first set have a region B_y different from the probes of the second set.

33. The method of claim 30, wherein the probes of one set are associated with the same label, said label unique from the probes of the other set.

34. A method to analyze nucleic acids comprising:

(a) hybridizing a first set of detectably labeled oligonucleotide probes to an array of target nucleic acids under reaction conditions optimized for multiple consecutive hybridization to full-match sequences;

(b) hybridizing a second set of detectably labeled oligonucleotide probes to the array under reaction conditions optimized for multiple consecutive hybridization to full-match sequences;

(c) ligating at least two detectably labeled probes that are hybridized to nucleic acids on the array; and

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

35. The method of claim 34, wherein each probe within a set has a same region B_y, and wherein the probes of the first set each have a region B_y different from that of the second set.

36. The method of claim 34, wherein the probes of one set are associated with the same label, said label unique from the probes of the other set.

37. The method of claim 34, wherein each probe within a set has the same detectable label.

38. The method of claim 34, further comprising:

(e) collecting multiple FRET signals; and

(f) comparing the collection to a pattern indicative of a known nucleic acid from an organism.

39. 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.

40. A method of amplifying a double-stranded target nucleic acid on a random array of nucleic acids, comprising the steps of:

(a) binding an invader oligonucleotide to one end of the target nucleic acid;

(b) hybridizing a first primer oligonucleotide to a single-stranded site on the target nucleic acid;

(c) initiating primer extension and displacement of one nucleic acid strand by a strand displacement polymerase;

(d) hybridizing a seconds primer oligonucleotide to a single stranded site of the target nucleic acid on the opposite strand to the single-stranded site in step (b);

(e) creating a new double stranded nucleic acid using the strand displacement polymerase; and

(f) repeating steps (a)-(e).

41. The method of claim 40, wherein the invader oligonucleotide comprises modifications that increase the binding affinity of the oligonucleotide to the target nucleic acid compared with the affinity of an unmodified oligonucleotide.

42. A method of amplifying a target DNA on a random array of nucleic acids, comprising the steps of:

(a) providing an invader oligonucleotide and a first primer complementary to a portion of the invader oligonucleotide;

(b) binding the invader oligonucleotide to one end of the target DNA;

(c) hybridizing the first primer oligonucleotide to a single-stranded site on the target nucleic acid;

(d) initiating primer extension and displacement of one strand of the DNA using a strand displacement polymerase;

(e) hybridizing a second primer oligonucleotide to a single-stranded site of the target DNA on the opposite strand to the single-stranded site in step (c);

(f) creating a new copy of the target DNA using the polymerase; and

(g) repeating steps (b) through (f).

43. The method of claim 42, wherein the strand displacement enzyme is Klenow fragment polymerase.

44. A method of isothermal amplification of a double-stranded target nucleic acid on a random array of nucleic acids, comprising the steps of:

(a) binding an invader oligonucleotide to the 5′

end of the target nucleic acid;

(b) hybridizing a first primer oligonucleotide to a single-stranded site on the target nucleic acid;

(c) initiating primer extension and displacement of one nucleic acid strand by a strand displacement polymerase;

(d) hybridizing a second primer oligonucleotide to a single stranded site of the target nucleic acid on the opposite strand to the single-stranded site in step (b);

(e) creating a new double stranded nucleic acid using the strand displacement polymerase; and

(f) repeating steps (a)-(e).

45. The method of claim 44, wherein the first primer is complementary to a portion of the invader oligonucleotide.

46. The method of claim 44, wherein the invader oligonucleotide comprises modifications that increase the binding affinity of the oligonucleotide to the target nucleic acid compared with the affinity of an unmodified oligonucleotide.

47. A method of isothermal amplification of a double-stranded target nucleic acid on a random array of nucleic acids, comprising the steps of:

(a) adding a double stranded nucleic acid fragment with low duplex stability to the 5′

end of the target nucleic acid;

(b) binding an invader oligonucleotide to the 5′

end of the target nucleic acid;

(c) hybridizing a first primer oligonucleotide to a single-stranded site on the target nucleic acid;

(d) initiating primer extension and displacement of one nucleic acid strand by a strand displacement polymerase;

(e) hybridizing a second primer oligonucleotide to a single stranded site of the target nucleic acid on the opposite strand to the single-stranded site in step (c);

(f) creating a new double stranded nucleic acid using the strand displacement polymerase; and

(g) repeating steps (b)-(f).

48. The method of claim 47, wherein the invader oligonucleotide comprises modifications that increase the binding affinity of the oligonucleotide to the target nucleic acid compared with the affinity of an unmodified oligonucleotide.

49. A method to analyze nucleic acids comprising:

(a) providing a random array of target nucleic acids, wherein the target nucleic acids are generated from multiple sources, and wherein each nucleic acid is tagged with an identifier of the particular source of the nucleic acid;

(b) hybridizing a population of oligonucleotide probes comprising the same region B_y to the array of target nucleic acids under reaction conditions optimized for hybridization to full-match sequences,

(c) collecting a signal produced by hybridization of oligonucleotide probes to the target nucleic acids on the array; and

(d) analyzing said signal;

wherein nucleic acids from a particular source are recognized by the assigned tag sequence.

50. The method of claim 49, wherein the reaction conditions are optimized for multiple consecutive hybridizations to full-match sequences.

51. A method of sequencing a plurality of nucleic acids from multiple sample sources, the method comprising:

(a) providing a random array of nucleic acids, wherein the nucleic acids are generated from multiple sources, and wherein each nucleic acid is tagged with identifier of the particular source of each nucleic acid;

(b) hybridizing a first set of detectably-labeled oligonucleotide probes to a random array of nucleic acids generated from the target nucleic acid under reaction conditions optimized for hybridization to full-match sequences;

(c) hybridizing a second set of detectably-labeled oligonucleotide probes to said target nucleic acids on the array under reaction conditions optimized for hybridization to full-match sequences;

(d) ligating at least one probe from each set that are hybridized to said target nucleic acids on the array; and

(e) detecting and analyzing said ligated probes;

wherein a sequence of each nucleic acid is identified by probe sequences that hybridize to the target molecule, and wherein nucleic acids from a particular source are recognized by the assigned tag sequence.

52. The method of claim 51, wherein the reaction conditions are optimized for multiple consecutive hybridizations to full-match sequences.

53. An assay for detecting a sequence in a target DNA fragment, comprising:

(a) providing a support comprising a plurality of target DNA fragments attached thereto;

(b) hybridizing two or more probes to target DNA fragments on said support;

(c) ligating the probes on said target nucleic acid that are hybridized to adjacent sites on a target DNA fragment to form a ligated probe pair;

(d) removing any probes that are not ligated to another probe on a target DNA fragment; and

(e) detecting ligated probe pairs hybridized to the target DNA fragment;

wherein detection of a ligated probe pair on a target DNA fragment is indicative of a sequence in the a target DNA fragment.

54. The assay of claim 53, wherein the assay is repeated to provide consecutive transient hybridizations, wherein the consecutive hybridizations result in multiple ligated probe pairs on the target DNA fragments.

55. An assay for detecting a sequence in a target DNA fragment, comprising:

(a) providing a support comprising a plurality of target DNA fragments attached thereto;

(b) hybridizing one or more probes to target DNA fragments, wherein said probes comprise a donor fluorophore for fluorescence resonance energy transfer (FRET) detection;

(c) hybridizing one or more probes to target DNA fragments, wherein said probes comprise a acceptor fluorophore for FRET detection;

(d) ligating probes on said target nucleic acid that are hybridized to adjacent sites on a target DNA fragment to form a ligated probe pair;

(e) removing any probes that are not ligated to another probe on a target DNA fragment; and

(f) detecting a fluorescence resonance energy transfer (FRET) signal between said ligated probes;

wherein detection of a FRET signal generated by a ligated probe pair on a target DNA fragment is indicative of a sequence in the a target DNA fragment.

56. The assay of claim 55, wherein the assay is repeated to provide consecutive transient hybridizations, wherein the consecutive hybridizations result in multiple ligated probe pairs on the target DNA fragments.

57. A sequence identification system, comprising:

(a) A random array of single molecule DNA fragments;

(b) A set of fluorescently tagged probe pairs from informative probe pools; and

(c) a camera for detection of hybridization/ligation events on the array;

wherein the system is designed to allowing testing of ligation of a given number of informative bases using multiple different probes capable of ligation with probes from the informative probe pools when hybridized contiguously on a target polynucleotide.

58. The system of claim 57, wherein the camera is a sensitive mega pixel CCD camera.

59. The system of claim 57, wherein DNA fragments are arrayed at a density of about 1 molecule per CCD pixel.