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

• US 20030175750A1
• Filed: 10/15/2002
• Published: 09/18/2003
• Est. Priority Date: 03/19/1999
• Status: Abandoned Application

• Current Assignee: Francis Barany, George Barany, Joseph Day, Nancy E. Witowski, Norman P. Gerry, Robert P. Hammer
• Original Assignee: Francis Barany, George Barany, Joseph Day, Nancy E. Witowski, Norman P. Gerry, Robert P. Hammer
• Inventors: Gerry, Norman P., Witowski, Nancy E., Day, Joseph, Barany, George, Barany, Francis, Hammer, Robert P.
• Application Number:
  US10/272,152
• Publication Number:
  US 20030175750A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/00326 Movement by rotation
• CPC Class Codes: B01J 2219/00416 Vacuum
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00729 Peptide nucleic acids [PNA]
• 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 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• 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: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/08 : Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• 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 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. The ligation phase can be preceded by an amplification process. The present invention also relates to a kit for practicing this method, a method of forming arrays on solid supports, and the supports themselves.

Claims

1. 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 comprising:

providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences;

providing a plurality of oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion and a detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample;

providing a ligase,

blending the sample, the plurality of oligonucleotide probe sets, and the ligase to form a mixture;

subjecting the mixture to one or more ligase detection reaction cycles comprising a denaturation treatment, wherein any hybridized oligonucleotides are separated from the target nucleotide sequences, and a hybridization treatment, wherein the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligated product sequence containing (a) the addressable array-specific portion, (b) the target-specific portions connected together, and (c) the detectable reporter label, and, wherein the oligonucleotide probe sets may hybridize to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment;

providing a solid support having a porous surface and different capture oligonucleotides immobilized at particular sites, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions;

contacting the mixture, after said subjecting, with the solid support under conditions effective to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the solid support at the site with the complementary capture oligonucleotide; and

detecting the reporter labels of ligated product sequences captured to the solid support at particular sites, thereby indicating the presence of one or more target nucleotide sequences in the sample.

2. A method according to claim 1, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, have a mismatch at a base at the ligation junction which interferes with such ligation.

3. A method according to claim 2, wherein the mismatch is at the 3′

base at the ligation junction.

4. A method according to claim 1, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, there is a mismatch at a base adjacent to a base at the ligation junction which interferes with such ligation.

5. A method according to claim 4, wherein the mismatch is at the base adjacent to the 3′

base at the ligation junction.

6. A method according to claim 1, wherein the sample potentially contains unknown amounts of one or more of a plurality of target sequences with a plurality of sequence differences, said method further comprising:

quantifying, after said detecting, the amount of the target nucleotide sequences in the sample by comparing the amount of captured ligated product sequences generated from the sample with a calibration curve of captured ligated product sequences generated from samples with known amounts of the, target nucleotide sequence.

7. A method according to claim 1, wherein the sample potentially contains unknown amounts of one or more of a plurality of target nucleotide sequences with a plurality of sequence differences, said method further comprising:

providing a known amount of one or more marker target nucleotide sequences;

providing a plurality of marker-specific oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and an addressable array-specific portion complementary to capture oligonucleotides on the solid support, and (b) a second oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and a detectable reporter label, wherein the oligonucleotide probes in a particular marker-specific oligonucleotide set are suitable for ligation together when hybridized adjacent to one another, on a corresponding marker target nucleotide sequence, but, when hybridized to any other nucleotide sequence present in the sample or added marker sequences, there is a mismatch which interferes with such ligation, wherein said blending comprises blending the sample, the marker target nucleotide sequences, the plurality of oligonucleotide probe sets, the plurality of marker-specific oligonucleotide probe sets, and the ligase to form a mixture;

detecting the reporter labels of the ligated marker-specific oligonucleotide sets captured on the solid support at particular sites, thereby indicating the presence of one or more marker target nucleotide sequences in the sample; and

quantifying the amount of target nucleotide sequences in the sample by comparing the amount of captured ligated product generated from the known amount of marker target nucleotide sequences with the amount of captured other ligated product.

8. A method according to claim 7, wherein the one or more marker target nucleotide sequences differ from the target nucleotide sequences in the sample at one or more single nucleotide positions.

9. A method according to claim 8, wherein the oligonucleotide probe sets and the marker-specific oligonucleotide probe sets form a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probe sets of each group, the first oligonucleotide probes have a common target-specific portion, and the second oligonucleotide probes have a differing target-specific portion which hybridize to a given allele or a marker nucleotide sequence in a base-specific manner.

10. A method according to claim 8, wherein the oligonucleotide probe sets and the marker-specific oligonucleotide probe sets form a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probe have differing target-specific portions, which hybridize to a given allele or a marker nucleotide sequence in a base-specific manner.

11. A method according to claim 1, wherein the sample potentially contains unknown amounts of two or more of a plurality of target nucleotide sequences with a plurality of sequence differences, said method further comprising:

quantifying, after said detecting, the relative amount of each of the plurality of target nucleotide sequences in the sample by comparing the relative amount of captured ligated product sequences generated by each of the plurality of target sequences within the sample, thereby providing a quantitative measure of the relative level of two or more target nucleotide sequences in the sample.

12. A method according to claim 1, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished with oligonucleotide probe sets having oligonucleotide probes with target-specific portions which overlap.

13. A method according to claim 1, wherein the target-specific portions of the oligonucleotide probe sets have substantially the same melting temperature so that they hybridize to target nucleotide sequences under similar hybridization conditions.

14. A method according to claim 1, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group, captured on the solid support at different sites, are detected, thereby indicating a presence, in the sample of one or more allele at one or more nucleotide position in one or more target nucleotide sequences.

15. A method according to claim 14, wherein the oligonucleotide probes in a given set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probe has a mismatch at a base at the ligation junction which interferes with such ligation.

16. A method according to claim 14, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished with oligonucleotide probe groups having oligonucleotide probes with target-specific portions which overlap.

17. A method according to claim 16, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, there is a mismatch at a base at the ligation junction which interferes with such ligation.

18. A method according to claim 1, wherein multiple allele differences consisting of insertions, deletions, microsatellite repeats, translocations, or other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets in a single target nucleotide sequence or multiple allele differences consisting of insertions, deletions, microsatellite repeats, translocations, or other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets in multiple target nucleotide sequences are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences selected from the group consisting of insertions, deletions, microsatellite repeats, translocations, and other DNA rearrangements at one or more nucleotide positions which requite overlapping oligonucleotide probe sets, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group, captured on the solid support at different sites, are detected, thereby indicating a presence, in the sample, of one or more allele differences selected from the group consisting of insertions, deletions, microsatellite repeats, translocations, and other DNA rearrangements in one or more target nucleotide sequences.

19. A method according to claim 18, wherein the oligonucleotide probe sets are designed for distinguishing multiple allele differences selected from the group consisting of insertions, deletions, and microsatellite repeats, at one or more nucleotide positions which require overlapping oligonucleotide probe sets, wherein, the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion, and the first oligonucleotide probes have differing target-specific portions which contain repetitive sequences of different lengths to hybridize to a given allele in a base-specific manner.

20. A method according to claim 1, wherein a low abundance of multiple allele differences at multiple adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, in the presence of an excess of normal sequence, or a low abundance of multiple allele differences at multiple nucleotide positions which require overlapping oligonucleotide probe sets in multiple target nucleotide sequences, in the presence of an excess of normal sequence, are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein one or more sets within a group share common second oligonucleotide probes and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele excluding the normal allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group captured on the solid support at different sites, are detected, thereby indicating a presence, in the sample, of one or more low abundance alleles at one or more nucleotide positions in one or more target nucleotide sequences.

21. A method according to claim 20, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide. sequence present in the sample, the first oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.

22. A method according to claim 20, wherein a low abundance of multiple allele differences at multiple adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, in the presence of an excess of normal sequence, or a low abundance of multiple allele differences at multiple nucleotide positions which require overlapping oligonucleotide probe sets in multiple target nucleotide sequences, in the presence of an excess of normal sequence, are quantified in a sample, said method further comprising:

providing a known amount of one or more marker target nucleotide sequences;

providing a plurality of marker-specific oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe having a target-specific portion complementary to the marker target nucleotide sequence and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and a detectable reporter label, wherein the oligonucleotide probes in a particular marker-specific oligonucleotide set are suitable for ligation together when hybridized adjacent to one another on a corresponding marker target nucleotide sequence, but, when hybridized to any other nucleotide sequence present in the sample or added marker sequences, have a mismatch which interferes with such ligation;

providing a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets or marker-specific oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, including marker nucleotide sequences, wherein one or more sets within a group share a common second oligonucleotide probe and the first oligonucleotide probes have different target-specific probe portions which hybridize to a given allele or a marker nucleotide sequence excluding the normal allele, in a base-specific manner, wherein said blending comprises blending the sample, the marker target nucleotide sequences, the plurality of oligonucleotide probe sets, the plurality of marker-specific oligonucleotide probe sets, and the ligase to form a mixture;

detecting the reporter labels of the ligated marker-specific oligonucleotide sets captured on the solid support at particular sites, thereby indicating the presence of one or more marker target nucleotide sequences in the sample; and

quantifying the amount of target nucleotide sequences in the sample by comparing the amount of captured ligated products generated from the known amount of marker target nucleotide sequences with the amount of other captured ligated product generated from the low abundance unknown sample.

23. A method according to claim 22, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence under selected 5 conditions due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.

24. A method according to claim 1, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of each group, the first oligonucleotide probes have a common target-specific portion and the second oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, different reporter labels of ligated product sequences of each group captured on the solid support at particular sites are detected, thereby indicating a presence, in the sample, of one or more allele at one or more nucleotide positions in one or more target nucleotide sequences.

25. A method according to claim 24, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.

26. A method according to claim 24, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished, the oligonucleotide probe groups containing oligonucleotide probes with target-specific portions which overlap.

27. A method according to claim 26, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the second oligonucleotide probe has a mismatch at a base at the ligation junction which interferes with such ligation.

28. A method according to claim 1, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of different groups, the second oligonucleotide probes have a common target-specific portion or the first oligonucleotide probes have a common target-specific portion, wherein, in said detecting, the one of a plurality of labeled ligated product sequences of each group captured on the solid support at particular sites are detected, thereby indicating a presence of one or more allele at one or more nucleotide positions in one or more target nucleotide sequences in the sample.

29. A method according to claim 28, wherein the oligonucleotide probes in a given set are suitable for ligation together at ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first or second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.

30. A method according to claim 28, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a target nucleotide sequence or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequence are distinguished, the oligonucleotide probe groups containing probes with target-specific portions which overlap.

31. A method according to claim 30, wherein oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first or second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.

32. A method according to claim 29, wherein all possible single-base mutations for a single codon in a single target nucleotide sequence, all possible single-base mutations for multiple codons in a single target nucleotide sequence, and all possible single-base mutations for multiple codons in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing all possible single-base mutations for a single codon, wherein, in the oligonucleotide probes of each group, the second oligonucleotide probes differ only in their 5′

bases at their ligation junction and contain different reporter labels, the first oligonucleotide probes differ only in their 3′

bases at their ligation junction and contain different addressable array-specific portions, or the first oligonucleotide probes differ only in their 3′

bases adjacent to the base at the ligation junction and contain different addressable array-specific portions.

33. A method according to claim 29, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at the 3′

base at the ligation junction or the base adjacent the base at the ligation junction or the second oligonucleotide probes have a mismatch at the 5′

base at the ligation junction which interferes with such ligation.

34. A method according to claim 33, wherein all possible single-base mutations for a single codon in a single target nucleotide sequence, or all possible single-base mutations for two or more adjacent codons, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished, the oligonucleotide probe groups containing oligonucleotide probes with target-specific portions which overlap.

35. A method according to claim 1, wherein the denaturation treatment is at a temperature of about 80°

-105°

C.

36. A method according to claim 1, wherein each cycle, comprising a denaturation treatment and a hybridization treatment, is from about 30 seconds to about five minutes long.

37. A method according to claim 1, wherein said subjecting is repeated for 2 to 50 cycles.

38. A method according to claim 1, wherein total time for said subjecting is 1 to 250 minutes.

39. A method according to claim 1, wherein the ligase is selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, Thermus sp. AK16 ligase, Aquifex aeolicus ligase, Thermotoga maritima ligase, and Pyrococcus ligase.

40. A method according to claim 1, wherein the detectable reporter label is selected from the group consisting of chromophores, fluorescent moieties, enzymes, antigens, heavy metals, magnetic probes, dyes, phosphorescent groups, radioactive materials, chemiluminescent moieties, and electrochemical detecting moieties.

41. A method according to claim 1, wherein the target-specific portions of the oligonucleotide probes each have a hybridization temperature of 40-85°

C.

42. A method according to claim 1, wherein the target-specific portions of the oligonucleotide probes are 20 to 28 nucleotides long.

43. A method according to claim 1, wherein the mixture further includes a carrier DNA.

44. A method according to claim 1, wherein said subjecting achieves, for a particular oligonucleotide probe set, a rate of formation of ligated product sequences that are mismatched at the site where the oligonucleotide probes for a particular oligonucleotide probe set are ligated which is less than 0.005 of the rate of formation of matched ligated product sequences for the particular oligonucleotide probe set.

45. A method according to claim 1 further comprising:

amplifying the target nucleotide sequences in the sample prior to said blending.

46. A method according to claim 45, wherein said amplifying is carried out by subjecting the sample to a polymerase-based amplifying procedure.

47. A method according to claim 45, wherein said polymerase-based amplifying procedure is carried out with DNA polymerase.

48. A method according to claim 1, wherein the solid support is made from a material selected from the group consisting of plastic, ceramic, metal, resin, gel, glass, silicon, and composites thereof.

49. A method according to claim 1, wherein said detecting comprises:

scanning the solid support at the particular sites and identifying if ligation of the oligonucleotide probe sets occurred and

correlating identified ligation to a presence or absence of the target nucleotide sequences.

50. A method according to claim 1, wherein the plurality of capture oligonucleotides each have different nucleotide sequences.

51. A method according to claim 50, wherein each capture oligonucleotide differs from its adjacent capture oligonucleotide on the array by at least one out of every four of the total number of nucleotides when the oligonucleotides are aligned at one end with one another without internal insertion or deletion.

52. A method according to claim 50, wherein each capture oligonucleotide has adjacent capture oligonucleotides separated from adjacent capture oligonucleotides by barrier oligonucleotides to which ligated oligonucleotide probe sets will not hybridize during said contacting.

53. A method according to claim 1, wherein the oligonucleotide probe sets hybridize to the target nucleotide sequences at temperatures which are less than that at which the capture oligonucleotides hybridize to the addressable array-specific portion of oligonucleotide probe sets.

54. A method according to claim 1 further comprising:

treating the mixture chemically or enzymatically, after said subjecting the mixture to a series of ligase detection reaction cycles, to destroy unligated oligonucleotide probes.

55. A method according to claim 54, wherein said treating is carried out with an exonuclease.

56. A method according to claim 1 further comprising:

removing oligonucleotides bound to the capture oligonucleotides to permit reuse of the solid support with immobilized capture oligonucleotides.

57. A method according to claim 1, wherein the solid support includes different capture oligonucleotides immobilized at different sites with different capture oligonucleotides being complementary to different addressable array-specific portions, whereby different oligonucleotide probe sets are captured and detected at different sites on the solid support.

58. A method according to claim 1, wherein the solid support includes identical capture oligonucleotides immobilized on the solid support with the capture oligonucleotides being complementary to all the addressable array-specific portions and the labels attached to the oligonucleotide probe sets being different, whereby the different oligonucleotide probe sets are detected and distinguished by the different labels.

59. A method according to claim 1, wherein the porous surface is a hydrophilic polymer composed of combinations of acrylamide with functional monomers containing carboxylate, aldehyde, or amino groups.

60. A method according to claim 59, wherein the functional monomer is acrylic acid.

61. A method according to claim 59, wherein the functional monomer is glycerol monomethacrylate.

62. A method according to claim 59, wherein the hydrophilic polymer is cross-link-ed at a level of less than 50:

1.

63. A method according to claim 62, wherein the hydrophilic polymer is cross-link-ed at a level of less than 500:

1.

64. An array of oligonucleotides on a solid support comprising:

a solid support having a porous surface and an array of positions each suitable for attachment of an oligonucleotide;

a linker or support suitable for coupling an oligonucleotide to the solid support attached to the solid support at each of the array positions;

and an array of oligonucleotides on the solid support with at least some of the array positions being occupied by oligonucleotides having greater than sixteen nucleotides.

65. An array according to claim 64, wherein different oligonucleotides are attached at different array positions on the solid support to detect different nucleic acids.

66. An array according to claim 64, wherein the solid support is made from a material selected from the group consisting of plastic, ceramic, metal, resin, gel, glass, silicon, and composites thereof.

67. An array according to claim 64, wherein the solid support has an array of positions with oligonucleotides attached to the array of positions.

68. An array according to claim 64, wherein the porous surface is a hydrophilic polymer composed of combinations of acrylamide with functional monomers containing carboxylate, aldehyde, or amino groups.

69. An array according to claim 68, wherein the functional monomer is acrylic acid.

70. An array according to claim 68, wherein the functional monomer is glycerol monomethacrylate.

71. An array according to claim 68, wherein the hydrophilic polymer is cross-linked at a level of less than 50:

1.

72. Am array according to claim 68, wherein the hydrophilic polymer is cross-linked at a level of less than 500:

1.

73. A kit for identifying one or more of a plurality of sequences differing by single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences comprising:

a ligase;

a plurality oligonucleotide probe sets, each characterized by (a) a first oligonucleotide probe, having a target sequence-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target sequence-specific portion and detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybrided adjacent to one another on a respective target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence, present in the sample; and

a solid support with a porous surface and capture oligonucleotides immobilized at particular sites, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions.

74. A kit according to claim 73, wherein the ligase is selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, Thermus sp. AK16 ligase, Aquifex aeolicus ligase, Thermotoga maritima ligase, and Pyrococcus ligase.

75. A kit according to claim 73 further comprising:

amplification primers suitable for preliminary amplification of the target nucleotide sequences and

a polymerase.

76. A kit according to claim 73, wherein the solid support includes different capture oligonucleotides immobilized at different particular sites with different capture oligonucleotides being complementary to different addressable array-specific portions, whereby different oligonucleotide probe sets are hybridized and detected at different sites on the solid support.

77. A kit according to claim 73, wherein the solid support includes identical capture oligonucleotides immobilized on the solid support with the capture oligonucleotides complementary to all the addressable array-specific portions and the labels attached to the oligonucleotide probe sets being different, whereby the oligonucleotide probe sets are detected and distinguished by the different labels.

78. A kit according to claim 73, wherein the oligonucleotide probe sets and the capture oligonucleotides are configured so that the oligonucleotide probe sets hybridize, respectively, to the target nucleotide sequences at temperatures which are less than that at which the capture oligonucleotides hybridize to the addressable array-specific portions of the oligonucleotide probes sets.

79. 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 comprising:

providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences;

providing a plurality of oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion and a detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample;

providing a ligase,

blending the sample, the plurality of oligonucleotide probe sets, and the ligase to form a mixture;

subjecting the mixture to one or more ligase detection reaction cycles comprising a denaturation treatment, wherein any hybridized oligonucleotides are separated from the target nucleotide sequences, and a hybridization treatment, wherein the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligated product sequence containing (a) the addressable array-specific portion, (b) the target-specific portions connected together, and (c) the detectable reporter label, and, wherein the oligonucleotide probe sets may hybridize to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment;

providing a solid support with different capture oligonucleotides immobilized at particular sites, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions;

contacting the mixture, after said subjecting, with the solid support under conditions effective to mask negative charges and to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the solid support at the site with the complementary capture oligonucleotide; and

detecting the reporter labels of ligated product sequences captured to the solid support at particular sites, thereby indicating the presence of one or more target nucleotide sequences in the sample.

80. A method according to claim 79, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, have a mismatch at a base at the ligation junction which interferes with such ligation.

81. A method according to claim 80, wherein the mismatch is at the 3′

base at the ligation junction.

82. A method according to claim 80, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, there is a mismatch at a base adjacent to a base at the ligation junction which interferes with such ligation.

83. A method according to claim 82, wherein the mismatch is at the base adjacent to the 3′

base at the ligation junction.

84. A method according to claim 79, wherein the sample potentially contains unknown amounts of one or more of a plurality of target sequences with a plurality of sequence differences, said method further comprising:

quantifying, after said detecting, the amount of the target nucleotide sequences in the sample by comparing the amount of captured ligated product sequences generated from the sample with a calibration curve of captured ligated product sequences generated from samples with known amounts of the target nucleotide sequence.

85. A method according to claim 79, wherein the sample potentially contains unknown amounts of one or more of a plurality of target nucleotide sequences with a plurality of sequence differences, said method further comprising:

providing a known amount of one or more marker target nucleotide sequences;

providing a plurality of marker-specific oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and an addressable array-specific portion complementary to capture oligonucleotides on the solid support, and (b) a second oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and a detectable reporter label, wherein the oligonucleotide probes in a particular marker-specific oligonucleotide set are suitable for ligation together when hybridized adjacent to one another on a corresponding marker target nucleotide sequence, but, when hybridize to any other nucleotide sequence present in the sample or added marker sequences, there is a mismatch which interferes with such ligation, wherein said blending comprises blending the sample, the marker target nucleotide sequences, the plurality of oligonucleotide probe sets, the plurality of marker-specific oligonucleotide probe sets, and the ligase to form a mixture;

detecting the reporter labels of the ligated marker-specific oligonucleotide sets captured on the solid support at particular sites, thereby indicating the presence of one or more marker target nucleotide sequences in the sample; and

quantifying the amount of target nucleotide sequences in the sample by comparing the amount of captured ligated product generated from the known amount of marker target nucleotide sequences with the amount of captured other ligated product.

86. A method according to claim 85, wherein the one or more marker target nucleotide sequences differ from the target nucleotide sequences in the sample at one or more single nucleotide positions.

87. A method according to claim 86, wherein the oligonucleotide probe sets and the marker-specific oligonucleotide probe sets form a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probe sets of each group, the first oligonucleotide probes have a common target-specific portion, and the second oligonucleotide probes have a differing target-specific portion which hybridize to a given allele or a marker nucleotide sequence in a base-specific manner.

88. A method according to claim 86, wherein the oligonucleotide probe sets and the marker-specific oligonucleotide probe sets form a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probe have differing target-specific portions, which hybridize to a given allele or a marker nucleotide sequence in a base-specific manner.

89. A method according to claim 79, wherein the sample potentially contains unknown amounts of two or more of a plurality of target nucleotide sequences with a plurality of sequence differences, said method further comprising:

quantifying, after said detecting, the relative amount of each of the plurality of target nucleotide sequences in the sample by comparing the relative amount of captured ligated product sequences generated by each of the plurality of target sequences within the sample, thereby providing a quantitative measure of the relative level of two or more target nucleotide sequences in the sample.

90. A method according to claim 79, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished with oligonucleotide probe sets having oligonucleotide probes with target-specific portions which overlap.

91. A method according to claim 79, wherein the target-specific portions of the oligonucleotide probe sets have substantially the same melting temperature so that they hybridize to target nucleotide sequences under similar hybridization conditions.

92. A method according to claim 79, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group, captured on the solid support at different sites, are detected, thereby indicating a presence, in the sample of one or more allele at one or more nucleotide position in one or more target nucleotide sequences.

93. A method according to claim 92, wherein the oligonucleotide probes in a given set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probe has a mismatch at a base at the ligation junction which interferes with such ligation.

94. A method according to claim 92, where multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished with oligonucleotide probe groups having oligonucleotide probes with target-specific portions which overlap.

95. A method according to claim 94, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, there is a mismatch at a base at the ligation junction which interferes with such ligation.

96. A method according to claim 79, wherein multiple allele differences consisting of insertions, deletions, microsatellite repeats, translocations, or other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets in a single target nucleotide sequence or multiple allele differences consisting of insertions, deletions, microsatellite repeats, translocations, or other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets in multiple target nucleotide sequences are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences selected from the group consisting of insertions, deletions, microsatellite repeats, translocations, and other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group, captured on the solid support at different sites, are detected, thereby indicating a presence, in the sample, of one or more allele differences selected from the group consisting of insertions, deletions, microsatellite repeats, translocations, and other DNA rearrangements in one or more target nucleotide sequences.

97. A method according to claim 79, wherein the oligonucleotide probe sets are designed for distinguishing multiple allele differences selected from the group consisting of insertions, deletions, and microsatellite repeats, at one or more nucleotide positions which require overlapping oligonucleotide probe sets, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion, and the first oligonucleotide probes have differing target-specific portions which contain repetitive sequences of different lengths to hybridize to a given allele in a base-specific manner.

98. A method according to claim 79, wherein a low abundance of multiple allele differences at multiple adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, in the presence of an excess of normal sequence, or a low abundance of multiple allele differences at multiple nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences, in the presence of an excess of normal sequence, are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein one or more sets within a group share common second oligonucleotide probes and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele excluding the normal allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group captured on the solid support at different sites, are detected, thereby indicating a presence, in the sample, of one or more low abundance alleles at one or more nucleotide positions in one or more target nucleotide sequences.

99. A method according to claim 98, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any-other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.

100. A method according to claim 99, wherein a low abundance of multiple allele differences at multiple adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, in the presence of an excess of normal sequence, or a low abundance of multiple allele differences at multiple nucleotide positions which require overlapping oligonucleotide probe sets in multiple target nucleotide sequences, in the presence of an excess of normal sequence, are quantified in a sample, said method further comprising:

providing a known amount of one or more marker target nucleotide sequences;

providing a plurality of marker-specific oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe having a target-specific portion complementary to the marker target nucleotide sequence and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and a detectable reporter label, wherein the oligonucleotide probes in a particular marker-specific oligonucleotide set are suitable for ligation together when hybridized adjacent to one another on a corresponding marker target nucleotide sequence, but, when hybridized to any other nucleotide sequence present in the sample or added marker sequences, have a mismatch which interferes with such ligation;

providing a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets or marker-specific oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, including marker nucleotide sequences, wherein one or more sets within a group share a common second oligonucleotide probe and the first oligonucleotide probes have different target-specific probe portions which hybridize to a given allele or a marker nucleotide sequence excluding the normal allele, in a base-specific manner, wherein said blending comprises blending the sample, the marker target nucleotide sequences, the plurality of oligonucleotide probe sets, the plurality of marker-specific oligonucleotide probe sets, and the ligase to form a mixture;

detecting the reporter labels of the ligated marker-specific oligonucleotide sets captured on the solid support at particular sites, thereby indicating the presence of one or more marker target nucleotide sequences in the sample; and

quantifying the amount of target nucleotide sequences in the sample by comparing the amount of captured ligated products generated from the known amount of marker target nucleotide sequences with the amount of other captured ligated product generated from the low abundance unknown sample.

101. A method according to claim 100, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence under selected conditions due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.

102. A method according to claim 79, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of each group, the first oligonucleotide probes have a common target-specific portion and the second oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, different reporter labels of ligated product sequences of each group captured on the solid support at particular sites are detected, thereby indicating a presence, in the sample, of one or more allele at one or more nucleotide positions in one or more target nucleotide sequences.

103. A method according to claim 102, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.

104. A method according to claim 102, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished, the oligonucleotide probe groups containing oligonucleotide probes with target-specific portions which overlap.

105. A method according to claim 104, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the second oligonucleotide probe has a mismatch at a base at the ligation junction which interferes, with such ligation.

106. A method according to claim 79, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of different groups, the second oligonucleotide probes have a common target-specific portion or the first oligonucleotide probes have a common target-specific portion, wherein, in said detecting, the one of a plurality of labeled ligated product sequences of each group captured on the solid support at particular sites are detected, thereby indicating a presence of one or more allele at one or more nucleotide positions in one or more target nucleotide sequences in the sample.

107. A method according to claim 106, wherein the oligonucleotide probes in a given set are suitable for ligation together at ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first or second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.

108. A method according to claim 106, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a target nucleotide sequence or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequence are distinguished, the oligonucleotide probe groups containing probes with target-specific portions which overlap.

109. A method according to claim 108, wherein oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first or second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.

110. A method according to claim 107, wherein all possible single-base mutations for a single codon in a single target nucleotide sequence, all possible single-base mutations for multiple codons in a single target nucleotide sequence, and all possible single-base mutations for multiple codons in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing all possible single-base mutations for a single codon, wherein, in the oligonucleotide probes of each group, the second oligonucleotide probes differ only in their 5′

bases at their ligation junction and contain different reporter labels, the first oligonucleotide probes differ only in their 3′

bases at their ligation junction and contain different addressable array-specific portions, or the first oligonucleotide probes differ only in their 3′

bases adjacent to the base at the ligation junction and contain different addressable array-specific portions.

111. A method according to claim 107, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at the 3′

base at the ligation junction or the 3′

base adjacent the base at the ligation junction or the second oligonucleotide probes have a mismatch at the 5′

base at the ligation junction which interferes with such ligation.

112. A method according to claim 111, wherein all possible single-base mutations for a single codon in a single target nucleotide sequence, or all possible single-base mutations for two or more adjacent codons, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished, the oligonucleotide probe groups containing oligonucleotide probes with target-specific portions which overlap.

113. A method according to claim 79, wherein the denaturation treatment is at a temperature of about 80-105°

C.

114. A method according to claim 79, wherein each cycle, comprising a denaturation treatment and a hybridization treatment, is from about 30 seconds to about five minutes long.

115. A method according to claim 79, wherein said subjecting is repeated for 2 to 50 cycles.

116. A method according to claim 79, wherein total time for said subjecting is 1 to 250 minutes.

117. A method according to claim 79, wherein the ligase is selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, Thermus sp. AK16 ligase, Aquifex aeolicus ligase, Thermotoga maritima ligase, and Pyrococcus ligase.

118. A method according to claim 79, wherein the detectable reporter label is selected from the group consisting of chromophores, fluorescent moieties, enzymes, antigens, heavy metals, magnetic probes, dyes, phosphorescent groups, radioactive materials, chemiluminescent moieties, and electrochemical detecting moieties.

119. A method according to claim 79, wherein the target-specific portions of the oligonucleotide probes each have a hybridization temperature of 40-85°

C.

120. A method according to claim 79, wherein the target-specific portions of the oligonucleotide probes are 20 to 28 nucleotides long.

121. A method according to claim 79, wherein the mixture further includes a carrier DNA.

122. A method according to claim 79, wherein said subjecting achieves, for a particular oligonucleotide probe set, a rate of formation of ligated product sequences that are mismatched at the site where the oligonucleotide probes for a particular oligonucleotide probe set are ligated which is less than 0.005 of the rate of formation of matched ligated product sequences for the particular oligonucleotide probe set.

123. A method according to claim 79 further comprising:

amplifying the target nucleotide sequences in the sample prior to said blending.

124. A method according to claim 123, wherein said amplifying is carried out by subjecting the sample to a polymerase-based amplifying procedure.

125. A method according to claim 123, wherein said polymerase-based amplifying procedure is carried out with DNA polymerase.

126. A method according to claim 79, wherein the solid support is made from a material selected from the group consisting of plastic, ceramic, metal, resin, gel, glass, silicon, and composites thereof.

127. A method according to claim 79, wherein said detecting comprises:

scanning the solid support at the particular sites and

identifying if litigation of the oligonucleotide probe sets occurred and correlating identified ligation to a presence or absence of the target nucleotide sequences.

128. A method according to claim 79, wherein the plurality of capture oligonucleotides each have different nucleotide sequences.

129. A method according to claim 128, wherein each capture oligonucleotide differs from its adjacent capture oligonucleotide on the array by at least one out of every four of the total number of nucleotides when the oligonucleotides are aligned at one end with one another without internal insertion or deletion.

130. A method according to claim 128, wherein each capture oligonucleotide has adjacent capture oligonucleotides separated from adjacent capture oligonucleotides by barrier oligonucleotides to which ligated oligonucleotide probe sets will not hybridize during said contacting.

131. A method according to claim 79, wherein the oligonucleotide probe sets hybridize to the target nucleotide sequences at temperatures which are less than that at which the capture oligonucleotides hybridize to the addressable array-specific portion of oligonucleotide probe sets.

132. A method according to claim 79 further comprising:

treating the mixture chemically or enzymatically, after said subjecting the mixture to a series of ligase detection reaction cycles, to destroy unligated oligonucleotide probes.

133. A method according to claim 132, wherein said treating is carried out with an exonuclease.

134. A method according to claim 79 further comprising:

removing oligonucleotides bound to the capture oligonucleotides to permit reuse of the solid support with immobilized capture oligonucleotides.

135. A method according to claim 79, wherein the solid support includes different capture oligonucleotides immobilized at different sites with different capture oligonucleotides being complementary to different addressable array-specific portions, whereby different oligonucleotide probe sets are captured and detected at different sites on the solid support.

136. A method according to claim 79, wherein the solid support includes identical capture oligonucleotides immobilized on the solid support with the capture oligonucleotides being complementary to all the addressable array-specific portions and the labels attached to the oligonucleotide probe sets being different, whereby the different oligonucleotide probe sets are detected and distinguished by the different labels.

137. A method according to claim 79, wherein the conditions effective to mask negative charges involves carrying out said contacting in the presence of a divalent cation-containing compound.

138. A method according to claim 137, wherein the divalent cation is selected from the group consisting of Mg⁺², Ca⁺², Mn⁺², and CO⁺².

139. A method according to claim 138, wherein the divalent cation is Mg⁺².

140. A method according to claim 79, wherein the conditions effective to mask negative charges involves carrying out said contacting at a pH at or below 6.0.

141. A method according to claim 79, wherein the conditions effective to mask negative charges involves capping free carboxylic acid groups with a neutralizing agent.

142. A method according to claim 141, wherein the neutralizing agent is selected from the group consisting of ethanolamine, diethanolamine, propanolamine, dipropanolamine, isopropanolamine, and diisopropanolamine.

143. A method according to claim 142, wherein the neutralizing agent is ethanolamine.

144. A method according to claim 79, wherein said contacting is carried out by mixing the mixture in the presence of the solid support.

145. A method according to claim 79, wherein the addressable array-specific portions are hybridized to the capture oligonucleotides, during said contacting, at a temperature of 60°

C. to 70°

C.

146. A method according to claim 79, wherein said detecting indicates the presence of ligated product in a ratio to unligated oligonucleotide probes of less than 1:

300.

147. A method according to claim 146, wherein said detecting indicates the presence of ligated product in a ratio to unligated oligonucleotide probes of less than 1:

900.

148. A method according to claim 147, wherein said detecting indicates the presence of ligated product in a ratio to unligated oligonucleotide probes of less than 1:

3000.

149. A method according to claim 148, wherein said detecting indicates the presence of ligated product in a ratio to unligated oligonucleotide probes of less than 1:

9000.

150. A method according to claim 79, wherein said detecting indicates the presence of a target nucleotide sequence, which differs from a non-target nucleotide sequence by a single base difference, in a ratio of the target nucleotide sequence to non-target nucleotide sequence of less than 1:

20.

151. A method according to claim 150, wherein said detecting indicates the presence of a target nucleotide sequence, which differs from a non-target nucleotide sequence by a single base difference, in a ratio of the target nucleotide sequence to non-target nucleotide sequence of less than 1:

50.

152. A method according to claim 151, wherein said detecting indicates the presence of a target nucleotide sequence, which differs from a non-target nucleotide sequence by a single base difference, in a ratio of the target nucleotide sequence to non-target nucleotide sequence of less than 1:

100.

153. A method according to claim 152, wherein said detecting indicates the presence of a target nucleotide sequence, which differs from a non-target nucleotide sequence by a single base difference, in a ratio of the target nucleotide sequence to non-target nucleotide sequence of less than 1:

200.