Method of designing addressable array for detection of nucleic acid sequence differences using ligase detection reaction

• US 20050142543A1
• Filed: 04/04/2001
• Published: 06/30/2005
• Est. Priority Date: 04/14/2000
• Status: Active Grant

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Zirvi, Monib, Barany, Francis, Kliman, Richard, Favis, Reyna, Gerry, Norman P.
• Granted Patent: US 7,455,965 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: H05K 999/99 dummy group

Abstract

The present invention is directed to a method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, where the plural capture oligonucleotide probes have melting temperatures within a narrow range. The first step of the method involves providing a first set of a plurality of tetramers of four nucleotides linked together, where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides. Groups of 2 to 4 of the tetramers from the first set are linked together to form a collection of multimer units. From the collection of multimer units, all multimer units formed from the same tetramer and all multimer units having a melting temperature in ° C. of less than 4 times the number of tetramers forming a multimer unit are removed to form a modified collection of multimer units. The modified collection of multimer units is arranged in a list in order of melting temperature. The order of the modified collection of multimer units is randomized in 2° C. increments of melting temperature.

Claims

1. A method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, wherein the plural capture oligonucleotide probes have melting temperatures within a narrow range, said method comprising:

providing a first set of a plurality of tetramers of four nucleotides linked together, wherein (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides;

linking groups of 2 to 4 of the tetramers from the first set together to form a collection of multimer units;

removing from the collection of multimer units all multimer units formed from the same tetramer and all multimer units having a melting temperature in °

C. of less than the 4 times the number of tetramers forming a multimer unit, to form a modified collection of multimer units;

arranging the modified collection of multimer units in a list in order of melting temperature;

randomizing, in 2°

C. increments of melting temperature, the order of the modified collection of multimer units;

dividing alternating multimer units in the list into first and second subcollections, each arranged in order of melting temperature;

inverting the order of the second subcollection;

linking in order the first collection of multimer units to the inverted second collection of multimer units in order to form a collection of double multimer units; and

removing from the collection of double multimer units those units (1) having a melting temperature in °

C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, to form a modified collection of double multimer units.

2. A method according to claim 1 further comprising:

placing the modified collection of double multimer units at positions on a support so that complementary oligonucleotides to be immobilized on the support can be captured at the positions.

3. A method according to claim 2, wherein the collection of double multimer units is shown in FIG. 26.

4. A method according to claim 2, wherein the collection of double multimer units is shown in FIG. 27.

5. A method according to claim 2, wherein the collection of double multimer units removed has a melting temperature in °

C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers.

6. A method according to claim 1, wherein the multimer units have 12 mers, the double multimer units have 24 mers, and the melting point of the double multimer units is 75-84°

C.

7. A method according to claim 1 further comprising:

reclaiming double multimer units having a melting temperature in °

C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers;

unlinking the reclaimed double multimers units to each form a pair of multimer units;

selecting multimer units with a melting temperature in °

C. of more than 11 times the number of tetramers and less than 17 times the number of tetramers; and

reintegrating the selected multimer units into said method.

8. A method according to claim 7, wherein the the method of claim 7 is repeated.

9. A method according to claim 1 further comprising:

making additional different sets of a plurality of tetramers by producing, base by base, circular permutations of the tetramers within the first set and complements thereof.

10. A method according to claim 1, wherein the set of tetramers is shown in Table 1 and complements thereof.

11. A method according to claim 10, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 1 and complements thereof.

12. A method according to claim 10, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 1 and complements thereof.

13. A method according to claim 10, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 1 and complements thereof.

14. A method according to claim 1, wherein the collection of double multimer units is shown in FIG. 26.

15. A method according to claim 1, wherein the modified collection of double multimer units is shown in FIG. 27.

16. A method according to claim 1, wherein the collection of double multimer units have a melting temperature in °

C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers.

17. An oligonucleotide array comprising:

a support and

a collection of double multimer unit oligonucleotides at different positions on the support so that complementary oligonucleotides to be immobilized on the support can be captured at the different positions, wherein the complementary oligonucleotides will hybridize, within a narrow temperature range of greater than 24°

C. with little mismatch, to members of the collection of double multimer unit oligonucleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it;

(1) oligonucleotides having a melting temperature in °

C. less than 12.5 times the number of tetramers and more than 14 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) multimer units with the same 4 tetramers linked together with or without interruption.

18. An oligonucleotide array according to claim 17, wherein the collection of double multimer units is shown in FIG. 26.

19. An oligonucleotide array according to claim 17, wherein the collection of double multimer units is shown in FIG. 27.

20. An oligonucleotide array according to claim 17, wherein the collection of double multimer units has a melting temperature in °

C. less than 12.5 times the number of tetramers and more than 14 times the number of tetramers.

21. An oligonucleotide array according to claim 17, wherein the double multimer units have 24 mers and the melting temperature of the double multimer units is 75-84°

C.

22. An oligonucleotide array according to claim 17, wherein the set of tetramers is shown in Table 6 and complements thereof.

23. An oligonucleotide array according to claim 17, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 6 and complements thereof.

24. A oligonucleotide array according to claim 17, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 6 and complements thereof.

25. An oligonucleotide array according to claim 17, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 6 and complements thereof.

26. 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 support with different capture oligonucleotides immobilized at different positions, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides, wherein oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of more than 4 times the number of tetramers in the multimer unit with little mismatch, to the capture oligonuncleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it;

(1) oligonucleotides having a melting temperature in °

C. of 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, to form a modified collection of double multimer units;

contacting the mixture, after said subjecting, with the 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 support at the site with the complementary capture oligonucleotide; and

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

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, have a mismatch at a base at the ligation junction which interferes with such ligation.

28. A method according to claim 27, wherein the mismatch is at the 3′

base at the ligation junction.

29. 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, there is a mismatch at a base adjacent to a base at the ligation junction which interferes with such ligation.

30. A method according to claim 29, wherein the mismatch is at the base adjacent to the 3′

base at the ligation junction.

31. A method according to claim 26, 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.

32. A method according to claim 26, 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 sequence;

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 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 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.

33. A method according to claim 31, 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.

34. A method according to claim 33, 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.

35. A method according to claim 33, 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 probes have differing target-specific portions, which hybridize to a given allele or a marker nucleotide sequence in a base-specific manner.

36. A method according to claim 26, 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.

37. A method according to claim 26, 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.

38. A method according to claim 26, 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 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.

39. A method according to claim 38, 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.

40. A method according to claim 38, 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.

41. A method according to claim 40, 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.

42. A method according to claim 26, 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 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.

43. A method according to claim 42, 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.

44. A method according to claim 26, 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 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.

45. A method according to claim 44, 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.

46. A method according to claim 44, 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 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.

47. A method according to claim 46, 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.

48. A method according to claim 26, 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 support at particular sites are detected, thereby indicating a presence, in the sample, of one or more alleles at one or more nucleotide positions in one or more target nucleotide sequences.

49. A method according to claim 48, 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.

50. A method according to claim 48, 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.

51. A method according to claim 50, 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.

52. A method according to claim 26, 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 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.

53. A method according to claim 52, 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 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.

54. A method according to claim 52, 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 sequences, are distinguished, the oligonucleotide probe groups containing probes with target-specific portions which overlap.

55. A method according to claim 54, 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.

56. A method according to claim 53, 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.

57. A method according to claim 53, 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.

58. A method according to claim 57, 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.

59. A method according to claim 26, wherein the denaturation treatment is at a temperature of about 80°

-105°

C.

60. A method according to claim 26, wherein each cycle, comprising a denaturation treatment and a hybridization treatment, is from about 30 seconds to about five minutes long.

61. A method according to claim 26, wherein said subjecting is repeated for 2 to 50 cycles.

62. A method according to claim 26, wherein total time for said subjecting is 1 to 250 minutes.

63. A method according to claim 26, wherein the ligase is selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, and Pyrococcus ligase.

64. A method according to claim 26, 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.

65. A method according to claim 26, wherein the target-specific portions of the oligonucleotide probes each have a hybridization temperature of 20-85°

C.

66. A method according to claim 26, wherein the target-specific portions of the oligonucleotide probes are 20 to 28 nucleotides long.

67. A method according to claim 26, wherein the mixture further includes a carrier DNA.

68. A method according to claim 26 further comprising:

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

69. A method according to claim 68, wherein said amplifying is carried out by subjecting the sample to a polymerase-based amplifying procedure.

70. A method according to claim 68, wherein said polymerase-based amplifying procedure is carried out with DNA polymerase.

71. A method according to claim 68, when rein said polymerase-based amplifying procedure is carried out with reverse transcriptase.

72. A method according to claim 68, wherein said polymerase-based amplifying procedure is carried out with RNA polymerase.

73. A method according to claim 68, wherein said amplifying is carried out by subjecting the target nucleotide sequences in the sample to a ligase chain reaction process.

74. A method according to claim 26, wherein the oligonucleotide probe sets are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, peptide nucleic acids, modified peptide nucleic acids, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof.

75. A method according to claim 26, wherein said method is used to detect infectious diseases caused by bacterial, viral, parasitic, and fungal infectious agents.

76. A method according to claim 75, wherein the infectious disease is caused by a bacteria selected from the group consisting of Escherichia coli Salmonella, Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, Yersinia, Francisella, Pasteurella, Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcus aureus, Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria, Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea, Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis, Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponema palladium, Borrelia burgdorferi, Borrelia recurrentis, Rickettsial pathogens, Nocardia, and Acitnomycetes.

77. A method according to claim 75, wherein the infectious disease is caused by a fungal infectious agent selected from the group consisting of Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccicioides brasiliensis, Candida albicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrix schenckii, Chromomycosis, and Maduromycosis.

78. A method according to claim 75, wherein the infectious disease is caused by a viral infectious agent selected from the group consisting of human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses (e.g., Hepatitis B Virus and Hepatitis C Virus), Epstein-Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, and reo viruses.

79. A method according to claim 75, wherein the infectious disease is caused by a parasitic infectious agent selected from the group consisting of Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonas spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necator americanis.

80. A method according to claim 26, wherein said method is used to detect genetic diseases.

81. A method according to claim 80, wherein the genetic disease is selected from the group consisting of 21 hydroxylase deficiency, cystic fibrosis, Fragile X Syndrome, Turner Syndrome, Duchenne Muscular Dystrophy, Down Syndrome, heart disease, single gene diseases, HLA typing, phenylketonuria, sickle cell anemia, Tay-Sachs Syndrome, thalassemia, Klinefelter'"'"'s Syndrome, Huntington'"'"'s Disease, autoimmune diseases, lipidosis, obesity defects, hemophilia, inborn errors in metabolism, and diabetes.

82. A method according to claim 26, wherein said method is used to detect cancer involving oncogenes, tumor suppressor genes, or genes involved in DNA amplification, replication, recombination, or repair.

83. A method according to claim 82, wherein the cancer is associated with a gene selected from the group consisting of BRCA1 gene, p53 gene, Familial polyposis coli, Her2/Neu amplification, Bcr/Ab1, K-ras gene, human papillomavirus Types 16 and 18, leukemia, colon cancer, breast cancer, lung cancer, prostate cancer, brain tumors, central nervous system tumors, bladder tumors, melanomas, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian carcinomas, ENT tumors, and loss of heterozygosity.

84. A method according to claim 26, wherein said method is used for environmental monitoring, forensics, and food and feed industry monitoring.

85. A method according to claim 26, wherein said detecting comprises:

scanning the 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.

86. A method according to claim 85, wherein said scanning is carried out by scanning electron microscopy, electron microscopy, confocal microscopy, charge-coupled device, scanning tunneling electron microscopy, infrared microscopy, atomic force microscopy, electrical conductance, and fluorescent or phosphor imaging.

87. A method according to claim 85, wherein said correlating is carried out with a computer.

88. A method according to claim 26, wherein said contacting the mixture with the support is at a temperature of 45-90°

C. and for a time period of up to 60 minutes.

89. A method according to claim 26, wherein some of the plurality of capture oligonucleotides have identical nucleotide sequences and different labels are used for some different target nucleotide sequence.

90. A method according to claim 26, wherein the plurality of capture oligonucleotides each have different nucleotide sequences.

91. A method according to claim 89, 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.

92. A method according to claim 26, 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.

93. A method according to claim 26 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.

94. A method according to claim 93, wherein said treating is carried out with an exonuclease.

95. A method according to claim 26 further comprising:

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

96. A method according to claim 26, wherein the 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 support.

97. A method according to claim 26, wherein the support includes identical capture oligonucleotides immobilized on the 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.

98. A method according to claim 26, wherein the collection of double multimer units is shown in FIG. 26.

99. A method according to claim 26, wherein the collection of double multimer units is shown in FIG. 27.

100. A method according to claim 26, wherein the collection of double multimer units removed has a melting temperature in °

C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers.

101. A method according to claim 26, wherein the double multimer units have 24 mers and the melting point of the double multimer units is 75-84°

C.

102. A method according to claim 26, wherein the set of tetramers is shown in Table 1 and complements thereof.

103. A method according to claim 26, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 1 and complements thereof.

104. A method according to claim 26, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 1 and complements thereof.

105. A method according to claim 26, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 1 and complements thereof.

106. 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 support with different capture oligonucleotides immobilized at different positions, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides, wherein oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of greater than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it;

(1) oligonucleotides having a melting temperature in °

C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions.

107. A kit according to claim 106, wherein the mismatch of oligonucleotide probe sets to nucleotide sequences other than their respective target nucleotide sequences is at a base at a ligation junction at which the oligonucleotide probe of each set ligate together when hybridized to their respective target nucleotide sequences.

108. A kit according to claim 106, wherein the mismatch is on the oligonucleotide probe of the oligonucleotide probe sets which have 3′

ends at the ligation junction.

109. A kit according to claim 106, wherein the mismatch of oligonucleotide probe sets to nucleotide sequences other than their respective target nucleotide sequence is at a base adjacent to a ligation junction at which the oligonucleotide probes of each set ligate together when hybridized to their respective target nucleotide sequences.

110. A kit according to claim 109, wherein the mismatch is on the oligonucleotide probe of the oligonucleotide probe sets which have 3′

ends at the ligation junction.

111. A kit according to claim 106, wherein the ligase is selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, and Pyrococcus ligase.

112. A kit according to claim 106 further comprising:

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

a polymerase.

113. A kit according to claim 106, wherein the 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 support.

114. A kit according to claim 106, wherein the support includes identical capture oligonucleotides immobilized on the 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.

115. A kit according to claim 106, 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.

116. A kit according to claim 106, wherein the collection of double multimer units is shown in FIG. 26.

117. A kit according to claim 106, wherein the collection of double multimer units is shown in FIG. 27.

118. A kit according to claim 106, wherein the collection of double multimer units removed has a melting temperature in °

C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers.

119. A kit according to claim 106, wherein the double multimer units have 24 mers and the melting point of the double multimer units is 75-84°

C.

120. A kit according to claim 106, wherein the set of tetramers is shown in Table 1 or complements thereof.

121. A kit according to claim 106, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 1 and complements thereof.

122. A kit according to claim 106, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 1 and complements thereof.

123. A kit according to claim 106, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 1 and complements thereof.

124. A method to avoid synthesizing ligase detection reaction oligonucleotides that will inappropriately cross-hybridize to capture oligonucleotides on a solid support comprising

comparing the ligase detection reaction oligonucleotides with the capture oligonucleotides and

identifying any capture oligonucleotides likely to cross-hybridize to the ligase detection reaction oligonucleotides.