DETECTION OF NUCLEIC ACID SEQUENCE DIFFERENCES USING THE LIGASE DETECTION REACTION WITH ADDRESSABLE ARRAYS

• US 20100173787A1
• Filed: 03/16/2010
• Published: 07/08/2010
• Est. Priority Date: 02/09/1996
• Status: Active Grant

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: ZIRVI, Monib, KEMPE, Maria, BLOK, Herman, BARANY, George, BARANY, Francis, HAMMER, Robert P.
• Granted Patent: US 7,892,747 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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. A method for identifying a plurality of target nucleotide sequences in a sample, said method comprising:

providing a sample potentially containing a plurality of target nucleotide sequences;

providing a plurality of composite oligonucleotide probes characterized by having a target-specific portion coupled to a zip code portion, wherein the zip code portions of the composite oligonucleotide probes comprise a nucleotide sequence having greater than sixteen nucleotides that differs for each target-specific portion and hybridizes to their complements under uniform hybridization conditions, wherein the nucleotide sequence of a complement to one zip code portion differs from the nucleotide sequence of a complement to another zip code portion, when aligned to each other, by at least 25% of the nucleotides, to minimize cross-hybridization of one zip code portion to a complement of another zip code portion;

providing one or more labels suitable for attachment to the composite oligonucleotide probes in the presence of its complementary target nucleotide sequence;

providing a plurality of secondary oligonucleotide probes, each comprising a nucleotide sequence complementary to that of a zip code portion of the composite oligonucleotide probes, wherein each zip code portion hybridizes to its complementary secondary oligonucleotide probe in a base-specific manner; and

detecting and distinguishing labeled products resulting from the hybridization of the target specific portions of the composite oligonucleotide probes to complementary target nucleotide sequences, if present, in the sample, and hybridization of the zip code portions of the composite oligonucleotide probes to their respective complementary secondary oligonucleotide probes, thereby identifying the presence of one or more target nucleotide sequences in the sample.

2. A method according to claim 1, wherein the plurality of secondary oligonucleotide probes are coupled to a solid support.

3. A method according to claim 1, wherein the plurality of secondary oligonucleotide probes are coupled to beads.

4. A method according to claim 2, wherein different secondary oligonucleotide probes are coupled to the solid support at different positions to detect different target nucleotide sequences.

5. A method according to claim 1, wherein the plurality of composite oligonucleotide probes are coupled to a solid support.

6. A method according to claim 1, wherein the plurality of composite oligonucleotide probes are coupled to beads.

7. A method according to claim 5, wherein different composite oligonucleotide probes are coupled to the solid support at different positions to detect different target nucleotide sequences.

8. A method according to claim 1 further comprising:

amplifying the target nucleotide sequences in the sample.

9. A method according to claim 8, wherein said amplifying is carried out by subjecting the sample to a polymerase-based amplifying procedure.

10. A method according to claim 9, wherein said polymerase-based amplifying procedure is carried out with DNA polymerase.

11. A method according to claim 1 further comprising:

blending the sample, the one or more labels, and the plurality of composite oligonucleotide probes to form a blend and

subjecting the blend to one or more hybridization treatments suitable for the target-specific portions of the composite oligonucleotide probes to hybridize in a base-specific manner to their respective target nucleotide sequences, if present in the sample, to form primary products, each product comprised of the composite oligonucleotide probe, the target nucleotide sequence hybridized in a base-specific manner to its respective composite oligonucleotide probe, and the one or more labels attached to the composite oligonucleotide probe only if its complementary target nucleotide sequence is present in the sample.

12. A method according to claim 11 further comprising:

providing a solid support with the plurality of secondary oligonucleotide probes attached at particular sites and

contacting the primary products with the solid support under uniform hybridization conditions suitable for hybridization in a base-specific manner of the zip code portions to their complementary secondary oligonucleotide probes under conditions that minimize cross hybridization between a zip code portion and a non-complementary secondary oligonucleotide probe, said detecting and distinguishing comprising;

detecting the labeled primary products captured on the solid support at particular sites, thereby indicating the presence of target nucleotide sequences in the sample.

13. The method according to claim 12, wherein each secondary oligonucleotide comprises 20-25 nucleotides.

14. The method according to claim 12, wherein the zip code portions hybridize to their complementary secondary oligonucleotides under highly stringent uniform hybridization conditions.

15. A method according to claim 11, wherein the blend comprises an enzyme capable of attaching the one or more labels to the composite oligonucleotide probes.

16. A method according to claim 15, wherein the enzyme is a ligase.

17. A method according to claim 11, wherein the one or more labels comprise oligonucleotide probes having a target-specific portion coupled to a reporter group and said subjecting comprises carrying out one or more ligase detection reaction cycles to form a ligation product comprising (a) the zip code portion, (b) the target-specific portions, and the reporter group if their respective target nucleotide sequence is present in the sample, said method further comprising:

providing a solid support with the plurality of secondary oligonucleotides immobilized at particular sites and

contacting the blend after carrying out said ligase detection reaction with the solid support under uniform hybridization conditions effective to hybridize the zip code portions of the ligation product to the complementary secondary oligonucleotides in a base-specific manner, thereby capturing the ligation products on the solid support at the site with the complementary secondary oligonucleotide, said detecting and distinguishing comprising;

detecting the ligation products captured on the solid support at particular sites, thereby indicating the presence of target nucleotide sequences in the sample.

18. The method according to claim 17, wherein each secondary oligonucleotide comprises 20-25 nucleotides.

19. The method according to claim 17, wherein the zip code portions of the ligation products hybridize to their complementary secondary oligonucleotides under highly stringent uniform hybridization conditions.

20. The method according to claim 17, wherein each ligase detection reaction cycle comprises a denaturation treatment and a hybridization treatment, each being from about 30 seconds to about five minutes long.

21. The method according to claim 20, wherein the denaturation treatment is at a temperature of about 80°

-105°

C.

22. The method according to claim 17, wherein the ligase detection reaction is carried out for 2 to 50 cycles.

23. The method according to claim 17, wherein total time for the ligase detection reaction is 1 to 250 minutes.

24. The method according to claim 17, wherein the target-specific portions of have a hybridization temperature of 20-85°

C.

25. The method according to claim 17, wherein the target-specific portions are 20 to 28 nucleotides long.

26. The method according to claim 17, wherein the ligase detection reaction is carried out with a ligase selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, and Pyrococcus ligase.