Detection of target nucleotide sequences using ligation assays with improved oligonucleotide probe pairs
A pair of oligonucleotide probes comprising a first oligonucleotide probe that comprises a first clamp section that is capable of hybridising to a second clamp section of a second oligonucleotide probe and a first target section that is capable of hybridising to a first section of a target DNA sequence to be detected, a second oligonucleotide probe that comprises a second clamp section that is capable of hybridising to the first clamp section of the first oligonucleotide probe and a second target section that is capable of hybridising to a second section of the target DNA sequence to be detected.
1. A pair of oligonucleotide probes (K) comprising:
(a) a first oligonucleotide probe (P 1 ) that comprises
(i) a first clamp segment (C 1 ), that is capable of hybridizing to a second clamp segment (C 2 ) of a second oligonucleotide probe (P 2 ), and
(ii) a first target segment (T 1 ) at its 3’ end that is capable of hybridizing to a first segment (S 1 ) of a target DNA sequence (D) to be detected;
(b) a second oligonucleotide probe (P 2 ) that comprises
(i) a second clamp segment (C 2 ), that is capable of hybridizing to C 1 , and
(ii) a second target segment (T 2 ) at it's 5’ end that is capable of hybridizing to a second segment (S 2 ) of D
wherein S 1 and S 2 are located essentially adjacent to one another in D, and wherein the 5’ end of S 1 is adjacent to the 3’ end of S 2 .
2. A pair of oligonucleotide probes according to claim 1 , wherein C 1 and C 2 when hybridized to each other have a melting temperature T mc which is higher than the melting temperature T mt of each of T 1 and T 2 when hybridized to a complementary DNA sequence.
3. A pair of oligonucleotide probes according to claim 2 , wherein the T mc of hybridized C 1 and C 2 is at least 1° C. higher than the highest T mt of hybridized T 1 and T 2 .
4. The pair of nucleotide probes of claim 3 wherein the T mc of hybridized C 1 and C 2 is at least 5° C. higher than the highest T mt of hybridized T 1 and T 2 .
5. The pair of nucleotide probes of claim 4 wherein the T mc of hybridized C 1 and C 2 is at least 10° C. higher than the highest T mt of T 1 and T 2 .
6. A pair of oligonucleotide probes according to claim 1 wherein the guanylic acid and cytidylic acid (GC) content of C 1 and C 2 ranges from more than 50% to 100%.
7. A pair of oligonucleotide probes according to claim 6 wherein the GC content of C 1 and C 2 is >60%.
8. A pair of oligonucleotide probes according to claim 7 wherein the GC content of C 1 and C 2 is >70%.
9. A pair of oligonucleotide probes according to claim 7 wherein the GC content of C 1 and C 2 is >80%.
10. A pair of oligonucleotide probes according to claim 7 wherein the GC content of C 1 and C 2 is between 90% and 100%.
11. A pair of oligonucleotide probes according to claim 1 , wherein C 1 and C 2 comprise, at least one G or C nucleotide more than the number of G or C nucleotides in T 1 or T 2 of the same length.
12. A pair of oligonucleotide probes according to claim 11 wherein C 1 and C 2 comprise at least two G or C nucleotides more than the number of G or C nucleotides in T 1 or T 2 of the same length.
13. A pair of oligonucleotide probes according to claim 12 wherein C 1 and C 2 comprise at least three G or C nucleotides more than the number of T 1 or T 2 of the same length.
14. A pair of oligonucleotide probes according to claim 13 wherein C 1 and C 2 comprise at least four G or C nucleotides more than the number of T 1 or T 2 of the same length.
15. A pair of oligonucleotide probes according to claim 14 wherein C 1 and C 2 comprise at least five G or C nucleotides more than the number of T 1 or T 2 of the same length.
16. A pair of oligonucleotide probes according to claim 1 , wherein the C 1 and/or C 2 comprises modified nucleotides that have an increased binding affinity compared to conventional adenylic acid (A), thymidilic acid (T), cytidylic acid (C) and guanylic acid (G).
17. A pair of oligonucleotide probes according to claim 1 , wherein the length of C 1 and/or C 2 is from 10 to 30 nucleotides.
18. A pair of oligonucleotide probes according to claim 17 , wherein the length of T 1 and T 2 is, independently, 15 to 30 nucleotides.
19. A pair of oligonucleotide probes according to claim 1 , wherein P 1 , P 2 or both includes at least one primer binding site designated B 1 or B 2 , respectively.
20. A pair of oligonucleotide probes according to claim 1 , wherein P 1 , and P 2 include at least one stuffer sequence designated R 1 or R 2 , respectively.
21. A pair of oligonucleotide probes according to claim 1 , wherein the each of T 1 and T 2 includes at least one allele-specific nucleotide.
22. A pair of oligonucleotide probes according to claim 21 , wherein the allele-specific nucleotide is located at the end of T 1 or of T 2 .
23. A set of oligonucleotide probes comprising the pair of oligonucleotides according to claim 21 and at least one additional probe (P 3 ) that includes a target segment T 3 having an additional allele specific nucleotide and wherein P 3 is distinct from P 1 and P 2 .
24. A pair of oligonucleotides probes according to claim 1 , wherein P 1 or P 2 comprises an additional region that cannot anneal to D, which additional region is located at the end of P 1 or P 2 at a position that corresponds to a junction site between S 1 and S 2 .
25. A pair of oligonucleotides probes according to claim 24 , wherein the additional region creates a cleavable site which site will be cleaved when exposed to a cleaving agent under conditions wherein cleavage can occur.
26. A group of oligonucleotides comprising at least two pairs of probes according to claim 1 , wherein the clamp segments C 1 and C 2 of each pair of probes are designed such that for each probe pair, the combination of C 1 and C 2 forms a unique combination within the group such that each probe can selectively hybridize to one other probe in the group.
27. A group of oligonucleotides according to claim 26 , wherein each C 1 and C 2 of each pair of probes further includes a unique sequence.
28. A method for the detection of a target nucleotide sequence (D) in a sample comprising the steps of:
(a) adding to the sample a pair (K) of probes according to claim 1 ;
(b) allowing the probes to hybridize to the target sequence;
(c) ligating T 1 and T 2 when they are located adjacent to one another when annealed to D to form ligation products; and
(d) detecting the presence or absence of any ligation products.
29. A method according to claim 28 , wherein the ligation products are amplified prior to the detecting step (d).
30. A method according to claim 28 , wherein D is amplified prior to hybridization of the probes.
31. A method according to claim 28 , wherein the sample includes more than one target nucleotide sequence designated D 1 . . . Dn, respectively, and wherein a group of more than one pair of oligonucleotide probes designated K 1 . . . Kn, respectively that correspond to, and are capable of binding to and detecting, D 1 . . . Dn, respectively, are added to the sample in step (a).
32. A method according to claim 31 wherein the clamp segments C 1 and C 2 of each pair of probes K 1 . . . Kn are designed such that for each probe pair K 1 . . . Kn, the combination of C 1 and C 2 forms a unique combination within the group such that for each probe pair K 1 . . . Kn, the probe P 1 can selectively hybridize to a unique single probe P 2 in the group and not to any other probe P 2 in the group.
33. A method according to claim 28 wherein each probe includes a unique sequence.
34. A method according to claim 28 wherein detection is based on measurement or assessment of nucleic acid length, sequence and/or mass.
35. A method according to claim 28 wherein the target sequence D is in a DNA or RNA molecule.
36. A method according to claim 35 wherein the DNA or RNA molecule is in the form of polyA+RNA, cDNA, genomic DNA, organelle DNA, a synthetic nucleic acid, a DNA library, a clone bank or any combination thereof.
37. A set of at least three oligonucleotides suitable for SNP genotyping, comprising:
(a) a first oligonucleotide probe (P 1 ) that comprises
(i) a first clamp segment (C 1 ) that is capable of hybridizing to a second clamp segment (C 2 ) of a second oligonucleotide probe (P 2 ), and
(ii) a first target segment (T 1 ) that is capable of hybridizing to a first segment (S 1 ) of a target DNA sequence (D) to be detected;
(b) a second oligonucleotide probe (P 2 ) that comprises
(i) a second clamp segment (C 2 ) that is capable of hybridizing to C 1 , and
(ii) a second target segment (T 2 ) that is capable of hybridizing to a second segment (S 2 ) of D;
(c) at least a third oligonucleotide probe (P 3 ) that comprises C 2 and T 2 ;
wherein P 2 and P 3 include an allele-specific nucleotide, located at the end of T 1 or T 2 ; and
wherein the allele-specific nucleotide of P 2 and P 3 corresponds to the alleles of the SNP to be detected; and
wherein P 2 and P 3 further include a stuffer segment that discriminates between amplified ligation products formed between P 1 and P 2 or P 1 and P 3 .
38. A kit useful in a method for detecting the presence, absence or amount of a target DNA sequence in a sample, the kit comprising at least one pair of probes as defined in claim 1 and one or more reagents to be used in said detecting method.
39. A kit useful in a method for detecting the presence, absence or amount of a target DNA sequence in a sample, the kit comprising at least one group of probes as defined in claim 26 and one or more reagents to be used in said detecting method.