Oligonucleotide arrays and their use for sorting, isolating, sequencing, and manipulating nucleic acids

• US 6,322,971 B1
• Filed: 09/30/1998
• Issued: 11/27/2001
• Est. Priority Date: 02/19/1992
• Status: Expired due to Term

• Current Assignee: University of Medicine and Dentistry of New Jersey (State of New Jersey)
• Original Assignee: The Public Health Research Institute of the City of New York, Inc.
• Inventors: Kramer, Fred Russell, C{acute over (h)}etverin, Alexander B.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Taylor, Janell E.
• Application Number:
  US09/164,249
• Time in Patent Office: 1,154 Days
• Field of Search: 435/6, 435/91.1, 435/810, 436/501, 436/808, 536/28, 536/29, 536/24.33, 536/25.3, 935/77
• 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 2219/00283 Reactor vessels with top opening
• CPC Class Codes: B01J 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00585 Parallel 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 2219/00637 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 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00662 Two-dimensional arrays within two-dimensional arrays
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 15/10 : Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6811 : Selection methods for production or design of target specific oligonucleotides or binding molecules
• 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 1/6853 : using modified primers or templates
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/131 : incorporating a restriction site
• CPC Class Codes: C12Q 2525/179 : incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 : incorporating an adaptor
• CPC Class Codes: C12Q 2531/107 : Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2563/179 : the label being a nucleic acid
• CPC Class Codes: C12Q 2565/519 : characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 : Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10S 435/81 : Packaged device or kit
• CPC Class Codes: Y10S 436/808 : Automated or kit

Abstract

Ligation methods for manipulating nucleic acid stands and oligonucleotides utilizing hybridization arrays of immobilized oligonucleotides. The oligonucleotide arrays may be plain or sectioned, comprehensive or non-comprehensive. The immobilized oligonucleotides may in some cases be binary oligonucleotides having constant as well as variable segments. Some embodiments include amplification of ligated products.

Claims

1. In a method wherein first and second nucleic acid strands are ligated to each other at their termini in order to form a recombinant product, the improvement comprising

a) providing an array of immobilized oligonucleotides comprising predetermined areas on a surface of a solid support, each area having immobilized thereon copies of an oligonucleotide,

b) hybridizing to said immobilized oligonucleotides first single-stranded terminal regions of first nucleic acid strands to be ligated, and

c) ligating to the hybridized first termini of said hybridized first strands to said second nucleic acid strands.

2. The method of claim 1 wherein the first nucleic acid strands hybridized to the immobilized oligonucleotides have different nucleotide sequences in different areas of the array.

3. The method according to claim 1 wherein the second nucleic acid strands being ligated to the hybridized first strands have different nucleotide sequences in different areas of the array.

4. The method according to claim 1, wherein the array is a comprehensive array.

5. The method acording to claim 1, wherein the oligonucleotides immobilized in each area are of the same length.

6. A method according to claim 1, wherein the oligonucleotides consist of the group consisting of deoxyribonucleotides, ribonucleotides, mixed deoxyribonucleotides and ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, and non-standard nucleotides.

7. The method according to claim 1, wherein the second nucleic acid strands are not also hybridized to the immobilized oligonucleotides.

8. The method according to claim 7 wherein the second nucleic acid strands are strands of double stranded nucleic acids.

9. The method according to claim 8 wherein the second nucleic acid strands are the same in every area of the array.

10. The method according to claim 8 wherein the array is a sectioned array and the recombinant product is amplified in a polymerase chain reaction amplification.

11. The method according to claim 8 wherein the first nucleic acid strands being hybridized to the immobilized oligonucleotides are contained in at least one mixture containing additional strands also containing first single-stranded terminal regions, and wherein the first single-stranded terminal regions of said additional strands differ from the first terminal regions of said first nucleic acid strands.

12. The method according claim 8 wherein the double stranded nucleic acids have one end adapted for ligation to blunt ends formed by hybridization of the first termini of the first nucleic acid strands to the immobilized oligonucleotides.

13. The method according to claim 12 wherein nonligating termini of the first nucleic acid strands and the double stranded second nucleic acid strands contain priming regions for amplification.

14. The method according to claim 12 wherein the immobilized oligonucleotides are RNA and the double stranded second nucleic acid strands are ligated to the immobilized oligonucleotides using RNA ligase prior to ligation of the first nucleic acid strands to the second nucleic acid strands.

15. The method according to claim 1 wherein the array is a sectioned array.

16. The method according to claim 1 wherein the second nucleic acid strands have second single-stranded terminal regions to be ligated to the first single-stranded regions of said first nucleic acid strands and wherein, prior to ligation, the second single-stranded terminal regions of the second nucleic acid strands are hybridized to the immobilized oligonucleotides in the array.

17. The method according to claim 16 wherein the first nucleic acid strands being hybridized to the immobilized oligonucleotides are contained in at least one mixture containing first additional strands also containing first single-stranded terminal regions, and wherein the first single-stranded terminal regions of said first additional strands differ from the first terminal regions of said first nucleic acid strands;

and, further, wherein the second nucleic acid strands being hybridized to the immobilized oligonucleotides are contained in at least one mixture containing second additional strands also containing second single-stranded terminal regions, and wherein the second single-stranded terminal regions of said second additional strands differ from the second terminal regions of said second nucleic acid strands.

18. The method according to claim 16 wherein the array is a binary oligonucleotide array whose immobilized oligonucleotides consist of a constant segment adjacent to a variable segment, and wherein the second nucleic acid strands are hybridized to the constant segment.

19. The method according to claim 16 wherein both the first and second nucleic acid strands contain priming regions at their non-ligating termini.

20. The method according to claim 19 wherein the array is a sectioned array and the recombinant product is amplified in a polymerase chain reaction.

21. A method of increasing the lengths of hybrids formed between nucleic acid strands and immobilized oligonucleotides in an array of immobilized oligonucleotides of different sequences, which hybrids have at least one single-stranded overhang that is a 3′

extension, comprising the steps of annealing to the overhang an extender oligonucleotide prepared by hybridizing a primer to the overhang and extending the primer with a DNA polymerase, and enzymatically ligating the extender to the hybrid if it is annealed immediately adjacent to the hybrid.

22. A process for ligating on an array of first, immobilized oligonucleotides of different sequences comprising the steps of

(a) incubating at least one nucleic acid strand with said immobilized oligonucleotides to form at least one hybrid having at least one single-stranded overhang comprising a 3′

extension,

(b) annealing to the overhangs a second oligonucleotide prepared by hybridizing a primer to the at least one overhang and extending the primer with a DNA polymerase, and

(c) enzymatically ligating the second oligonucleotides to that at least one hybrid if it is annealed immediately adjacent thereto, thereby forming at least one hybrid of increased length.

23. A method of increasing the lengths of hybrids formed between nucleic acid strands of undetermined sequence in a sample and immobilized oligonucleotides in an array of immobilized oligonucleotides of different sequences, which hybrids have at least one single-stranded overhang, comprising the steps of annealing an extender oligonucleotide to the overhang and enzymatically ligating it to the hybrid if it is annealed immediately adjacent to the hybrid.

24. The method of claim 23 wherein the overhang is a 3′

extension of the nucleic acid strands beyond the free 5′

ends of the immobilized nucleotides.

25. A method of increasing the lengths of hybrids formed between nucleic acid strands and immobilized oligonucleotides in an array of immobilized binary oligonucleotides having different sequences and having a constant segment adjacent to a free-end variable segment, which hybrids have at least one single-stranded overhang, comprising the steps of annealing to the overhang an extender oligonucleotide that is hybridized to, and covers at least a part of, said constant segment, and enzymatically ligating it to the hybrid if it is annealed immediately adjacent to the hybrid.

26. The method of claim 25 wherein the extender oligonucleotide is annealed to the immobilized oligonucleotide prior to formation of the nucleic acid strand-immobilized oligonucleotide hybrid.

27. The method of claim 25 further comprising the subsequent step of washing under stringent conditions in which only extended hybrids will not be washed away.

28. A target-dependent process for ligating on an array of first, immobilized oligonucleotides of different sequences comprising the steps of

(a) incubating a sample containing at least one nucleic acid strand of undetermined sequence with said immobilized oligonucleotides to form at least one hybrid having at least one single-stranded overhang,

(b) annealing second oligonucleotides to the overhangs, and

(c) enzymatically ligating the second oligonucleotides to the hybrids if they are annealed immediately adjacent thereto, thereby forming hybrids of increased length.

29. The process of claim 28 wherein step b) precedes step a).

30. The process of claim 28 further comprising the step of washing away strands not bound to the array by hybrids of increased length.

31. A method for introducing a plurality of site directed mutations into different copies of a parent nucleic acid strand on a sectioned oligonucleotide array using different-length partial copies of said parent strand, said copies corresponding to regions of the parent nucleic acid strand adjacent to the location of site directed mutations, comprising the steps of

a) separately ligating said partial copies to free termini of immobilized oligonucleotides in said oligonucleotide array to obtain mutated partial copies of said strand, said oligonucleotide array comprising an array of predetermined areas on the surface of said support, each area having therein a different immobilized oligonucleotide having a sequence adapted to introduce a sequence containing a site directed mutation to a partial copy added to that area;

and

b) using the mutated partial copies to generate nucleic acid strands that are full-length mutant copies of the parent strand containing the site directed mutations.

32. The method according to claim 31 wherein step b is accomplished by

(i) hybridizing a complementary copy of each mutated partial copy to a template having a sequence complementary to the remaining terminal portion of the parent nucleic acid strand which is not contained in said partial copy; and

(ii) carrying out a polymerase chain reaction, a ligation reaction or both a polymerase chain reaction and a ligation reaction to join the remaining terminal portion of the parent nucleic acid strand to the mutated partial copy.