Methods and apparatus for DNA sequencing and DNA identification

• US 6,270,961 B1
• Filed: 12/09/1994
• Issued: 08/07/2001
• Est. Priority Date: 04/01/1987
• Status: Expired due to Fees

• Current Assignee: Hyseq, Inc. (Arca Biopharma, Inc.)
• Original Assignee: Hyseq, Inc. (Arca Biopharma, Inc.)
• Inventors: Drmanac, Radoje
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/353,554
• Time in Patent Office: 2,433 Days
• Field of Search: 435/5, 435/6, 435/912, 436/518, 536/24.3, 536/24.31, 536/24.32, 536/24.33
• US Class Current: 435/6.12
• 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 2219/00369 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00504 Pins
• CPC Class Codes: B01J 2219/00513 Essentially linear supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

Sequencing by Hybridization (SBH) methods and apparatus employing subdivided filters for discrete multiple probe analysis of multiple samples may be used for DNA identification and for DNA sequencing. Partitioned filters are prepared. Samples are affixed to sections of partitioned filters and each sector is probed with a single probe or a multiplexed probe for hybridization scoring. Hybridization data is analyzed for probe complementarity, partial sequencing by SBH or complete sequencing by SBH.

Claims

1. A method of analyzing nucleic acids by hybridization, comprising the steps of:

contacting a first plurality of different nucleic acid segments disposed on a first sector of a substrate with a first hybridization oligonucleotide, wherein said first plurality of nucleic acids is arranged on said first sector in a spatially identifiable array, under conditions that discriminate between a perfect match and a single base mismatch in the hybridization of a member of said first array of nucleic acid segments to the informational portion of said first hybridization oligonucleotide;

contacting a second plurality of different nucleic acid segments disposed on a second sector of the substrate with a second hybridization oligonucleotide, wherein said second plurality of nucleic acids is arranged on said second sector in a spatially identifiable array, under conditions that discriminate between a perfect match and a single base mismatch in this hybridization of a member of said second array of nucleic acid segments to the informational portion of said second hybridization oligonucleotide,

wherein said second hybridization oligonucleotide differs in sequence from said first hybridization oligonucleotide;

wherein said first plurality of nucleic acid segments is not contacted with said second hybridization oligonucleotide;

detecting hybridization of a hybridization oligonucleotide to a nucleic acid segment; and

analyzing the result.

2. Thc method as recited in claim 1 wherein said first contacting step comprises the step of applying a plurality of contiguously hybridizing oligonucleotides.

3. The method as recited in claim 1 wherein said second contacting step comprises the step of applying a plurality of contiguously hybridizing oligonucleotides.

4. The method as recited in claim 2 further comprising the step of ligating at least two of said plurality of contiguously hybridizing oligonucleotides.

5. The method as recited in claim 3 further comprising the step of ligating at least two of said plurality of contiguously hybridizing oligonucleotides.

6. The method as recited in claim 1 wherein said first contacting step comprises the step of applying a plurality of competitively hybridizing oligonucleotides having overlapping nucleic acid sequences.

7. The method as recited in claim 1 wherein said second contacting step comprises the step of applying a plurality of competitively hybridizing oligonucleotides having overlapping nucleic acid sequences.

8. The method as recited in claim 1 wherein a least two of said first plurality of nucleic acid segments are disposed as a mixture.

9. The method as recited in claim 1 wherein a least two of said second plurality of nucleic acid segments are disposed as a mixture.

10. The method as recited in claim 1 further comprising the step of selecting deoxyribonucleotide oligonucleotides.

11. The method as recited in claim 1 further comprising the step of selecting ribonucleotide oligonucleotides.

12. The method as recited in claim 1 further comprising the step of multiplex labeling of oligonucleotides.

13. The method as recited in claim 1 further comprising the step of degrading a label on an unhybridized oligonucleotide.

14. The method as recited in claim 1 further comprising the step of stripping the substrate of oligonucleotides for reuse of said pluralities of nucleic acid segments.

37. The method according to either claim 1 or claim 23, wherein the nucleic acids arrayed on said sector are derived from a subject.

15. A method for analyzing a plurality of nucleic acids from a plurality of subjects, comprising the steps of:

providing a substrate comprising the nucleic acids, wherein the nucleic acids are disposed on a plurality of sectors on the substrate, and wherein different nucleic acids are arranged on each sector in a spatially identifiable array;

contacting the nucleic acids with a plurality of oligonucleotide probes under conditions that discriminate between a perfect match and a single base mismatch in the hybridization of a member of said plurality of nucleic acid segments to the informational portion of a member of said plurality of oligonucleotides,

wherein the different nucleic acids on a first sector are contacted with a first probe and the nucleic acids on a different second sector are contacted with a second probe,

wherein the second probe differs in sequence from the first probe and the first sector is not contacted with the second probe; and

determining which probes bind to the nucleic acids, thereby identifying single nucleotide polymorphisms among the plurality of samples.

16. The method of claim 15, wherein the subjects all share a particular phenotype.

17. The method of claim 16, wherein the nucleic acids represent a large number of genes from the subject.

18. The method of claim 17, further comprising the step of determining which mutations are shared by several subjects.

19. The method of claim 15, wherein the subjects are members of a species.

20. The method of claim 19, wherein the subjects have been exposed to a plurality of different environmental conditions.

21. The method of claim 15, wherein the subjects are members of a plurality of species.

22. The method of claim 21, wherein the nucleic acids include an evolutionarily conserved DNA sequence.

23. A method for analyzing a nucleic acid, comprising the steps of:

providing a substrate comprising the nucleic acid, wherein the nucleic acid is disposed on a plurality of sectors on the substrate, and wherein each sector comprises a spatially identifiable array of immobilized nucleic acids;

contacting the nucleic acid with a plurality of oligonucleotides under conditions that discriminate between a perfect match and a single base mismatch in the hybridization of the nucleic acid to the informational portion of a member of said plurality of oligonucleotides,

wherein the nucleic acid in a first sector of the substrate is contacted with a first oligonucleotide and the nucleic acid in a second sector of the substrate is contacted with a second oligonucleotide,

wherein the second oligonucleotide differs in sequence from the first oligonucleotide and the first sector is not contacted with the second oligonucleotide; and

determining which oligonucleotides bind to the nucleic acid.

24. The method of claim 23, wherein the plurality of oligonucleotides contains a first set of oligonucleotides having the formula N_XaB_YaN_Za wherein the B_Ya portions are capable of forming perfectly matched duplexes with a fragment of the nucleic acid and a second set of oligonucleotides having the formula N_XbB_YbN_Zb wherein the B_Yb portions are capable of forming mismatched duplexes with a fragment of the nucleic acid, wherein the fragment has a known sequence.

25. The method of claim 24, wherein the fragment is part of an STS.

26. The method of claim 24, wherein the fragment is part of an antibiotic resistance gene.

27. The method of claim 24, wherein the fragment is part of an HIV.

28. The method of claim 23, wherein the plurality of oligonucleotide contains a plurality of paired sets of first oligonucleotides having the formula N_XaB_YaN_Za wherein the B_Ya portions are capable of forming perfectly matched duplexes with a plurality of fragments in the nucleic acid and second oligonucleotides having the formula N_XbB_YbN_Zb wherein the B_Yb portions are capable of forming mismatched duplexes with a plurality of fragments in the nucleic acid, wherein the fragments have known sequences.

29. The method of claim 28, wherein the plurality of fragments are from an HIV.

30. The method of claim 24, wherein the fragment contains a single nucleotide polymorphism.

31. The method of claim 28, wherein the fragments contain a plurality of single nucleotide polymorphisms.

32. The method of claim 31, wherein each fragment has a different single nucleotide polymorphism.

33. The method of claim 28, wherein the fragment is part of an STS.

34. The method of claim 28, wherein the fragment is part of an antibiotic resistance gene.

35. The method of claim 24 wherein the amount of nucleic acid is determined by comparing the intensity of hybridization for several of the positive oligonucleotides with a sample containing the nucleic acid and with a control sample containing a known amount of DNA.

36. The method of claim 28, wherein the amount of nucleic acid is determined by comparing the intensity of hybridization for several of the first oligonucleotide with a sample containing the nucleic acid and with a control sample containing a known amount of DNA.