Random array DNA analysis by hybridization

• US 8,278,039 B2
• Filed: 10/31/2007
• Issued: 10/02/2012
• 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.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US11/981,797
• Publication Number:
  US 20090011416A1
• Time in Patent Office: 1,798 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 system for determining sequence information for a polynucleotide, comprising:

(a) an array of DNA molecules, wherein each DNA molecule comprises a fragment of said polynucleotide;

(b) a set of informative oligonucleotide probes, wherein each probe comprises a label and a nucleotide sequence comprising the formula N_xB_yN_z, wherein;

(i) each N is independently a degenerate base;

(ii) each B is independently an informative base;

(iii) x and z are each at least one; and

(iv) y is 2 to 20;

(c) a detector configured to detect labeled probes from said set once the probes have hybridized to DNA molecules in the array, and to send data indicating the labeled probes that have been detected to a computer processor; and

(d) a computer processor programmed to receive said data from the detector, and to process the data to obtain said sequence information for the polynucleotide.

2. The system of claim 1, wherein the detector is a megapixel CCD camera.

3. The system of claim 2, wherein said DNA molecules are arrayed at a density of about 1 molecule per CCD pixel.

4. The system of claim 1, wherein each DNA molecule comprises multiple copies of the respective fragment.

5. The system of claim 1, wherein y is 2 to 8.

6. The system of claim 1, wherein the labels are fluorescent labels.

7. The system of claim 1, wherein the DNA molecules on the array comprise overlapping fragments of an entire human genome.

8. The system of claim 1, wherein the DNA molecules are arrayed on a support in a grid pattern.

9. A system for determining sequence information for a polynucleotide, comprising:

(a) an array of DNA molecules, wherein each DNA molecule comprises a fragment of said polynucleotide;

(b) a first set of informative oligonucleotide probes, wherein each probe comprises a label and a nucleotide sequence comprising the formula N_xB_y or the formula B_yN_z, wherein;

(i) each N is independently a degenerate base;

(ii) each B is independently an informative base;

(iii) x and z are each at least one; and

(iv) y is 2 to 20;

(c) a detector configured to detect labeled probes from said set once the probes have hybridized to DNA molecules in the array, and to send data indicating the labeled probes that have been detected to a computer processor; and

(d) a computer processor programmed to receive said data from the detector, and to process the data to obtain said sequence information for the polynucleotide.

10. The system of claim 9, wherein each probe of the set comprises the formula N_xB_y.

11. The system of claim 9, wherein each probe of the set comprises the formula B_yN_z.

12. The system of claim 9, further comprising:

(e) a second set of oligonucleotides; and

(f) a ligase for ligating an oligonucleotide probe from the first set to an oligonucleotide from the second set when hybridized to adjacent sites on a DNA molecule on the array.

13. The system of claim 12, wherein the first set of probes is a universal probe set.

14. The system of claim 9, wherein x is 1 to 4.

15. The system of claim 9, wherein y is 2 to 8.

16. The system of claim 9, wherein y is 4 to 5.

17. The system of claim 9, wherein z is 1 to 4.

18. The system of claim 9, wherein the labels are fluorescent labels.

19. The system of claim 9, wherein each DNA molecule comprises multiple copies of the respective fragment.

20. The system of claim 9, wherein the DNA molecules on the array comprise overlapping fragments of an entire human genome.

21. The system of claim 9, wherein the DNA molecules are arrayed on a support in a grid pattern.

22. A product for determining sequence information for a polynucleotide, comprising:

(a) an array of DNA molecules, wherein each DNA molecule comprises a fragment of said polynucleotide; and

(b) oligonucleotide probes hybridized to a plurality of the DNA molecules on the array, said probes constituting a first set wherein each probe comprises a label and a nucleotide sequence comprising the formula N_xB_y or the formula B_yN_z, wherein;

(i) each N is independently a degenerate base;

(ii) each B is independently an informative base;

(iii) x and z are each at least one; and

(iv) y is 2 to 20.

23. The product of claim 22, wherein each probe of the first set comprises the formula N_xB_y.

24. The product of claim 22, wherein each probe of the first set comprises the formula B_yN_z.

25. The product of claim 22, further comprising a second set of oligonucleotides each hybridized to one of the DNA molecules at a position that is adjacent to an oligonucleotide probe of the first set.

26. The product of claim 25, wherein said oligonucleotides from the second set are each ligated to the adjacent probe from the first set.

27. The product of claim 22, wherein x is 1 to 4.

28. The product of claim 22, wherein y is 2 to 8.

29. The product of claim 22, wherein y is 4 to 5.

30. The product of claim 22, wherein z is 1 to 4.

31. The product of claim 22, wherein the labels are a fluorescent labels.

32. The product of claim 22 wherein each DNA molecule comprises multiple copies of the respective fragment.

33. The product of claim 22, wherein the DNA molecules on the array comprise overlapping fragments of an entire human genome.

34. The product of claim 22, wherein the DNA molecules are arrayed on a support in a grid pattern.

35. The product of claim 22, wherein probes in the first set comprise the formula N_xB_yN_z.

36. A system for determining sequence information for a polynucleotide, comprising:

(a) an array of DNA molecules, wherein each DNA molecule comprises a fragment of said polynucleotide;

(b) a first set of informative oligonucleotide probes, wherein each probe comprises a label and a nucleotide sequence comprising the formula N_xB_y or the formula B_yN_z, wherein;

(i) each N is independently a degenerate base;

(ii) each B is independently an informative base; and

(iii) x, y, and z are each at least one;

(c) a second set of oligonucleotide probes, each configured to bind to a site in a DNA molecule of the array adjacent to a probe from the first set;

(d) a detector configured to detect labeled probes from said set once the probes have hybridized to DNA molecules in the array, and to send data indicating the labeled probes that have been detected to a computer processor; and

(e) a computer processor programmed to receive said data from the detector, and to process the data to obtain said sequence information for the polynucleotide.

37. The system of claim 36, further comprising a ligase for ligating an oligonucleotide probe from the first set to an oligonucleotide from the second set when hybridized to adjacent sites on a DNA molecule on the array.

38. The system of claim 36, wherein probes in the first set comprise the formula N_xB_yN_z.

39. The system of claim 36, wherein y is 2 to 8.

40. The system of claim 36, wherein the DNA molecules on the array comprise overlapping fragments of an entire human genome.

41. A product for determining sequence information for a polynucleotide, comprising:

(a) an array of DNA molecules, wherein each DNA molecule comprises a fragment of said polynucleotide; and

(b) oligonucleotide probes hybridized to a plurality of the DNA molecules on the array, said probes constituting a first set wherein each probe comprises a label and a nucleotide sequence comprising the formula N_xB_y or the formula B_yN_z, wherein;

(i) each N is independently a degenerate base;

(ii) each B is independently an informative base;

(iii) x, y, and z are each at least one; and

(c) a second set of oligonucleotides each hybridized to a plurality of the DNA molecules at a position that is adjacent to an oligonucleotide probe of the first set.

42. The product of claim 41, wherein oligonucleotides from the second set that are hybridized to the DNA molecules are ligated to an adjacent probe from the first set.

43. The product of claim 41, wherein probes in the first set comprise the formula N_xB_yN_z.

44. The product of claim 41, wherein y is 2 to 8.

45. The product of claim 41, wherein the DNA molecules on the array comprise overlapping fragments of an entire human genome.