Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays

• US 8,703,928 B2
• Filed: 09/09/2011
• Issued: 04/22/2014
• Est. Priority Date: 02/09/1996
• Status: Expired due to Fees

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Barany, Francis, Barany, George, Hammer, Robert P., Kempe, Maria, Blok, Herman, Zirvi, Monib
• Primary Examiner(s): Martinell, James
• Application Number:
  US13/229,198
• Publication Number:
  US 20120071364A1
• Time in Patent Office: 956 Days
• Field of Search: None
• US Class Current: 536/24.3
• 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 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• 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 2219/00621 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 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00729 Peptide nucleic acids [PNA]
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• 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 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2537/143 : Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/125 : Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/501 : being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/514 : characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

The present invention describes a method for identifying one or more of a plurality of sequences differing by one or more single base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences. The method includes a ligation phase, a capture phase, and a detection phase. The ligation phase utilizes a ligation detection reaction between one oligonucleotide probe, which has a target sequence-specific portion and an addressable array-specific portion, and a second oligonucleotide probe, having a target sequence-specific portion and a detectable label. After the ligation phase, the capture phase is carried out by hybridizing the ligated oligonucleotide probes to a solid support with an array of immobilized capture oligonucleotides at least some of which are complementary to the addressable array-specific portion. Following completion of the capture phase, a detection phase is carried out to detect the labels of ligated oligonucleotide probes hybridized to the solid support.

Claims

1. An instrument comprising:

a solid support comprising at least 25 types of capture oligonucleotides immobilized on the solid support at an array of positions, wherein each type of capture oligonucleotide is greater than 16 nucleotides and differs in nucleotide sequence, when aligned to another type of capture oligonucleotide that is located on an adjacent position of said solid support, by at least 25%;

one or more nucleic acid molecules hybridized to complementary portions of the one or more capture oligonucleotides on said solid support, wherein the capture oligonucleotides hybridize to the complementary portions of the nucleic acid molecules under uniform hybridization conditions; and

an imager configured to detect the hybridized nucleic acid molecules.

2. The instrument of claim 1, wherein the solid support comprises a high-density array of capture oligonucleotides immobilized thereto.

3. The instrument of claim 1, wherein the solid support comprises 25 to 1200 types of capture oligonucleotides.

4. The instrument of claim 1, wherein the solid support comprises at least 1000 types of capture oligonucleotides.

5. The instrument of claim 1, wherein the one or more nucleic acid molecules each comprise an addressable array-specific portion comprising a nucleotide sequence that is complementary to the capture oligonucleotide sequence, and a target nucleotide portion comprising a nucleotide sequence that is the same as, or complementary to, a target nucleotide sequence.

6. The instrument of claim 5, wherein the target nucleotide portions of the nucleic acid molecules are greater than 16 nucleotides.

7. The instrument of claim 1, wherein the nucleic acid molecules comprise amplification products, or complements thereof, of one or more cellular target nucleic acid molecules.

8. The instrument of claim 1, wherein each capture oligonucleotide on the solid support comprises 20-25mers.

9. The instrument of claim 1, wherein each type of capture oligonucleotide differs in nucleotide sequence, when aligned to any other type of capture oligonucleotide of said solid support, by at least 25%.

10. The instrument of claim 1, wherein the imager is a fluorescence imager and the nucleic acid molecules hybridized to the one or more capture oligonucleotides each comprise a fluorescent label suitable for detection by the fluorescence imager.

11. An instrument comprising:

a solid support comprising at least 25 types of capture oligonucleotides immobilized on the solid support, wherein capture oligonucleotides at an array position on the solid support comprise the same nucleotide sequence and wherein each type of capture oligonucleotide is greater than 16 nucleotides and differs in nucleotide sequence, when aligned to another type of capture oligonucleotide that is located on an adjacent position of said solid support, by at least 25%;

one or more nucleic acid molecules hybridized to complementary portions of the one or more capture oligonucleotides on said solid support, wherein the capture oligonucleotides hybridize to the complementary portions of the nucleic acid molecules under uniform hybridization conditions and wherein the one or more nucleic acid molecules each comprise an addressable array-specific portion comprising a nucleotide sequence that is complementary to the capture oligonucleotide sequence, and a target nucleotide portion comprising a nucleotide sequence that is the same as, or complementary to, a target nucleotide sequence; and

a fluorescence imager configured to detect the hybridized nucleic acid molecules, wherein the nucleic acid molecules hybridized to the one or more capture oligonucleotides each comprise a fluorescent label suitable for detection by the fluorescence imager.

12. The instrument of claim 11, wherein the solid support comprises a high-density array of capture oligonucleotides immobilized thereto.

13. The instrument of claim 11, wherein the solid support comprises 25 to 1200 types of capture oligonucleotides.

14. The instrument of claim 11, wherein the solid support comprises at least 1000 types of capture oligonucleotides.

15. The instrument of claim 11, wherein the target nucleotide portions of the nucleic acid molecule are greater than 16 nucleotides.

16. The instrument of claim 11, wherein the nucleic acid molecules comprise amplification products, or complements thereof, of one or more cellular target nucleic acid molecules.

17. The instrument of claim 11, wherein two or more different hybridized nucleic acid molecules at an array position comprise different fluorescent labels.

18. The instrument of claim 17, wherein four different hybridized nucleic acid molecules at an array position comprise four different fluorescent labels.

19. The instrument of claim 11, wherein two different hybridized nucleic acid molecules at an array position comprise different fluorescent labels.

20. The instrument of claim 11, wherein two or more different hybridized nucleic acid molecules have the same fluorescent label and are discriminated by different addressable array-specific portions.

21. The instrument of claim 11, wherein each capture oligonucleotide on the solid support comprises 20-25mers.

22. The instrument of claim 11, wherein each type of capture oligonucleotide differs in nucleotide sequence, when aligned to any other type of capture oligonucleotide of the solid support, by at least 25%.