Method of detecting nucleic acids

• US 6,197,506 B1
• Filed: 04/08/1998
• Issued: 03/06/2001
• Est. Priority Date: 06/07/1989
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Solas, Dennis W., Dower, William J., Fodor, Stephen P. A.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US09/056,927
• Time in Patent Office: 1,063 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 506/3
• 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 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00434 Liquid crystal masks
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00621 : by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 : Covalent
• CPC Class Codes: B01J 2219/0063 : Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00644 : the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• 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 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07C 229/14 : to carbon atoms of carbon skeletons containing rings
• CPC Class Codes: C07C 229/16 : to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
• CPC Class Codes: C07D 263/44 : Two oxygen atoms
• CPC Class Codes: C07D 317/62 : with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
• CPC Class Codes: C07H 19/04 : Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/10 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 17/06 : attached to the carrier via a bridging agent
• CPC Class Codes: C07K 17/14 : Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 : for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 : Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/131 : Allele specific probes
• CPC Class Codes: C12Q 2563/149 : Particles, e.g. beads
• CPC Class Codes: C12Q 2565/626 : being a flow cytometer
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 15/1433 : using image recognition
• 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 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K
• CPC Class Codes: G03F 7/265 : Selective reaction with inorganic or organometallic reagents after image-wise exposure, e.g. silylation
• CPC Class Codes: G03F 7/38 : Treatment before imagewise removal, e.g. prebaking G03F7/265 takes precedence
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules

Abstract

The present invention provides methods and apparatus for sequencing, fingerprinting and mapping biological macromolecules, typically biological polymers. The methods make use of a plurality of sequence specific recognition reagents which can also be used for classification of biological samples, and to characterize their sources.

Claims

1. A method for distinguishing two nucleic acids samples comprising:

(a) providing a substrate having at least 100 different polynucleotides in known locations at a density of at least 50 different polynucleotides per square centimeter,

(b) hybridizing a first nucleic acid sample to the substrate to obtain a first hybridization pattern,

(c) hybridizing a second nucleic acid sample to the substrate to obtain a second hybridization pattern, and

(d) comparing the first hybridization pattern with the second hybridization pattern, thereby distinguishing the first nucleic acid sample from the second nucleic acid sample.

2. The method of claim 1, wherein at least part of the sequence of each of the polynucleotides is known.

3. The method of claim 1, wherein at least some of the polynucleotides have a length of 25 to 50 nucleotides.

4. The method of claim 1, wherein the difference is detected by comparing a first partial nucleotide sequence generated from the first hybridization pattern with a second partial nucleotide sequence generated from the second hybridization pattern.

24. The method of claim 1, wherein the substrate has at least 1000 different polynucleotides in known locations.

25. The method of claim 1, wherein the density is at least 300 different polynucleotides per square centimeter.

26. The method of claim 1, wherein the density is at least 3000 different polynucleotides per square centimeter.

5. A method for determining whether a test sample of nucleic acid is from an individual comprising:

(a) providing a reference sample of nucleic acid from the individual,

(b) providing a substrate having a plurality of different polynucleotides in known locations at a density of at least 50 different polynucleotides per square centimeter,

(c) applying the reference sample to the substrate to obtain a reference hybridization pattern,

(d) applying the test sample to the substrate to obtain a test hybridization pattern, and

(e) comparing the reference hybridization pattern with the test hybridization pattern to determine whether the test sample is from the individual.

27. The method of claim 5, wherein the substrate has at least 1000 different polynucleotides in known locations.

28. The method of claim 5, wherein the density is at least 300 different polynucleotides per square centimeter.

29. The method of claim 5, wherein the density is at least 3000 different polynucleotides per square centimeter.

6. A method for identifying a nucleic acid sample comprising:

(a) providing a substrate having a plurality of different polynucleotides in known locations at a density of at least different 50 polynucleotides per square centimeter,

(b) applying the nucleic acid sample to the substrate to obtain a hybridization pattern, and

(c) comparing the hybridization pattern with a reference database to identify the nucleic acid sample.

7. The method of claim 6, wherein at least part of the sequence of each of the polynucleotides is known.

8. The method of claim 6, wherein at least some of the polynucleotides have a length of 25 to 50 nucleotides.

9. The method of claim 6, wherein the hybridization pattern is analyzed to generate a partial nucleotide sequence for the nucleic acid sample, and the partial nucleotide sequence is compared with a reference nucleotide sequence in the reference database.

30. The method of claim 6, wherein the substrate has at least 1000 different polynucleotides in known locations.

31. The method of claim 6, wherein the density is at least 300 different polynucleotides per square centimeter.

32. The method of claim 6, wherein the density is at least 3000 different polynucleotides per square centimeter.

10. A method for identifying a sample of nucleic acid comprising:

(a) providing the sample,

(b) providing a substrate having a plurality of different polynucleotides in known locations at a density of at least different 50 polynucleotides per square centimeter,

(c) applying the sample to the substrate to obtain a hybridization pattern, and

(d) comparing the hybridization pattern to a reference database of a plurality of sources of nucleic acid, thereby identifying the source of the sample.

11. The method of claim 10, wherein at least part of the sequence of each of the polynucleotides is known.

12. The method of claim 10, wherein at least some of the polynucleotides have a length of 25 to 50 nucleotides.

13. The method of claim 10, wherein the hybridization pattern is analyzed to generate a partial nucleotide sequence, and the partial nucleotide sequence is compared with a reference nucleotide sequence in the reference database.

14. The method of claim 10, wherein the sample is from an individual, and the plurality of sources of nucleic acid are provided by potential relatives of the individual thereby identifying a genealogy of the individual.

15. The method of claim 10, wherein the sample is provided by an organism, and the sources of nucleic acid are provided by a plurality of known individuals, thereby identifying the organism as one of the known individuals.

33. The method of claim 10, wherein the substrate has at least 1000 different polynucleotides in known locations.

34. The method of claim 10, wherein the density is at least 300 different polynucleotides per square centimeter.

35. The method of claim 10, wherein the density is at least 3000 different polynucleotides per square centimeter.

16. A method for detecting genetic variation among different individuals comprising:

(a) providing a sample from each of a plurality of individuals, wherein the sample contains nucleic acid of the individual;

(b) providing a substrate having a plurality of polynucleotides in known locations at a density of at least 50 different polynucleotides per square centimeter;

(c) applying each sample to the substrate to obtain a hybridization pattern; and

(d) comparing the hybridization patterns obtained to detect genetic variation.

36. The method of claim 16, wherein the substrate has at least 1000 different polynucleotides in known locations.

37. The method of claim 16, wherein the density is at least 300 different polynucleotides per square centimeter.

38. The method of claim 16, wherein the density is at least 3000 different polynucleotides per square centimeter.

17. A method of detecting genetic variation related to a disease comprising:

(a) providing samples of nucleic acid from each of a plurality of individuals, wherein at least some individuals have a high genetic propensity for the disease and at least some other individuals have low genetic propensity for the disease;

(b) providing a substrate having a plurality of polynucleotides in known locations at a density of at least 50 different polynucleotides per square centimeter;

(c) applying each sample to the substrate to obtain a hybridization pattern; and

(d) correlating the hybridization patterns obtained to high genetic propensity for the disease.

39. The method of claim 17, wherein the substrate has at least 1000 different polynucleotides in known locations.

40. The method of claim 17, wherein the density is at least 300 different polynucleotides per square centimeter.

41. The method of claim 17, wherein the density is at least 3000 different polynucleotides per square centimeter.

18. A method for detecting a mutation in an individual comprising:

(a) providing a sample of nucleic acid from the individual;

(b) providing a substrate having a plurality of oligonucleotides in known locations at a density of at least different 50 oligonucleotides per square centimeter, wherein at least some oligonucleotides have mutant sequence and at least some other oligonucleotides have wild type sequence;

(c) applying each sample to the substrate to obtain a hybridization pattern; and

(d) detecting mutation in the individual by analysis of the hybridization pattern.

42. The method of claim 18, wherein the substrate has at least 1000 different oligonucleotides in known locations.

43. The method of claim 18, wherein the density is at least 300 different oligonucleotides per square centimeter.

44. The method of claim 18, wherein the density is at least 3000 different oligonucleotides per square centimeter.

19. A method for diagnosing an abnormal tissue comprising:

(a) providing a sample from the abnormal tissue, wherein the sample contains transcripts of the abnormal tissue,

(b) providing a substrate having a plurality of different polynucleotides in known locations at a density of at least different 50 polynucleotides per square centimeter,

(c) applying the sample to the substrate to obtain an expression pattern, and

(d) comparing the expression pattern to a reference database having expression patterns for a plurality of known abnormalities of tissue, thereby diagnosing the abnormal tissue.

45. The method of claim 19, wherein the substrate has at least 1000 different polynucleotides in known locations.

46. The method of claim 19, wherein the density is at least 300 different polynucleotides per square centimeter.

47. The method of claim 19, wherein the density is at least 3000 different polynucleotides per square centimeter.

20. A method for determining cellular composition of a tissue comprising:

(a) providing a sample from the tissue, wherein the sample contains nucleic acid of the tissue;

(b) providing a substrate having a plurality of polynucleotides in known locations at a density of at least different 50 polynucleotides per square centimeter;

(c) applying the sample to the substrate to obtain an expression pattern; and

(d) comparing the expression pattern to a reference database having expression patterns for a plurality of known cells, thereby determining the cellular composition of the tissue.

48. The method of claim 20, wherein the substrate has at least 1000 different polynucleotides in known locations.

49. The method of claim 20, wherein the density is at least 300 different polynucleotides per square centimeter.

50. The method of claim 20, wherein the density is at least 3000 different polynucleotides per square centimeter.

21. A method for identifying a microbe comprising:

(a) providing a sample from the microbe, wherein the sample contains nucleic acid of the microbe;

(b) providing a substrate having a plurality of polynucleotides in known locations at a density of at least different 50 polynucleotides per square centimeter;

(c) applying the sample to the substrate to obtain a hybridization pattern; and

(d) comparing the hybridization pattern with a reference database to identify the microbe.

51. The method of claim 21, wherein the substrate has at least 1000 different polynucleotides in known locations.

52. The method of claim 21, wherein the density is at least 300 different polynucleotides per square centimeter.

53. The method of claim 21, wherein the density is at least 3000 different polynucleotides per square centimeter.

22. A method for detecting genetic variation among different isolates of a microbe comprising:

(a) providing a sample from each of a plurality of isolates, wherein the sample contains nucleic acid of the isolate;

(b) providing a substrate having a plurality of polynucleotides in known locations at a density of at least 50 different polynucleotides per square centimeter;

(c) applying each sample to the substrate to obtain a hybridization pattern; and

(d) comparing the hybridization patterns obtained to detect genetic variation.

23. The method of claim 22, wherein the microbe is selected from the group consisting of protozoa, virus and bacteria.

54. The method of claim 22, wherein the substrate has at least 1000 different polynucleotides in known locations.

55. The method of claim 22, wherein the density is at least 300 different polynucleotides per square centimeter.

56. The method of claim 22, wherein the density is at least 3000 different polynucleotides per square centimeter.