Granted Patent B2

US 8,283,121 · App. 12/496,447 · Granted Oct 9, 2012

Assignee: Cornell Research Foundation, Inc.

Abstract

Claims (46)

1. A method for identifying one or more different target nucleotide sequences comprising:

providing a sample potentially containing one or more target nucleotide sequences comprising sequence differences;

providing one or more oligonucleotide probe sets, each probe set comprising (a) a first oligonucleotide probe comprising a target-specific portion, a further portion, and a 5′ upstream primer-specific portion and (b) a second oligonucleotide probe comprising a target-specific portion and a 3′ downstream primer-specific portion, wherein the first and second oligonucleotide probes in a particular set are suitable for ligation together when hybridized on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when the first and second oligonucleotide probes are hybridized to any other nucleotide sequence present in the sample;

providing a ligase;

blending the sample, the one or more oligonucleotide probe sets, and the ligase to form a ligase reaction mixture;

subjecting the ligase reaction mixture to one or more ligase reaction cycles to form a ligation product sequence comprising (a) the 5′ upstream primer specific portion, (b) the further portion, (c) the target-specific portions, and (d) the 3′ downstream primer-specific portion, when the respective target nucleotide sequence of the corresponding oligonucleotide probe set is present in the sample;

providing one or a plurality of oligonucleotide primer sets, each set comprising (a) an upstream primer and (b) a downstream primer;

providing a polymerase;

blending the ligase reaction mixture with the one or a plurality of oligonucleotide primer sets, and the polymerase to form a polymerase chain reaction mixture;

subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles to form extension products comprising the ligation product sequence and/or complements thereof; and

detecting the extension products to identify one or more target nucleotide sequences in the sample.

2. The method according to claim 1 , wherein the upstream primer is complementary to the 5′ upstream primer-specific portion of the ligation product sequence and the downstream primer is complementary to the 3′ downstream primer-specific portion of the ligation product sequence.

3. The method according to claim 1 , wherein the ligation product sequence of the oligonucleotide probes in each set produces an extension product of unique length, said method further comprising:

separating the extension products by size or electrophoretic mobility, wherein said detecting differentiates the extension products which differ in size.

4. The method according to claim 1 , wherein the further portion of the oligonucleotide probe in a particular probe set is configured so that the sequence of the ligation product sequence from each oligonucleotide probe set is unique and can be distinguished from other nucleic acids in the polymerase chain reaction mixture, said method further comprising:

providing a solid support with different capture oligonucleotides immobilized at different particular sites, wherein the capture oligonucleotides have nucleotide sequences complementary to the further portion of a given probe set and

contacting the polymerase chain reaction mixture, after said subjecting it to one or more polymerase chain reaction cycles, with the solid support under conditions effective to hybridize the extension products to the capture oligonucleotides in a base-specific manner, wherein said detecting indicates the presence of extension products captured using the further portions to identify one or more target nucleotide sequences in the sample.

5. The method according to claim 1 , wherein the relative amounts of two or more differing sequences are present in a sample in unknown amounts with a plurality of target nucleotide sequences being quantified and a set of oligonucleotide primers being useful in amplifying all the ligation product sequences formed by the oligonucleotide probe sets, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of two or more oligonucleotide probe sets, wherein oligonucleotide probe sets in the same group comprise the same 5′ upstream primer-specific portion and the same 3′ downstream primer-specific portion, said method further comprising:

quantifying the relative amount of the extension products, after said detecting and

comparing relative amounts of the extension products generated to provide a quantitative measure of the relative level of the two or more target nucleotide sequences in the sample.

6. The method according to claim 5 , wherein oligonucleotide probe sets in the same group comprise the same 5′ upstream primer-specific portion and the same 3′ downstream primer-specific portion, and the ligation product sequences of oligonucleotide probes in each particular set have a unique length so that they can be distinguished from other nucleic acids in the polymerase chain reaction mixture, said method further comprising:

separating the extension products by size or electrophoretic mobility, wherein said detecting is carried out by size differences in the extension products.

7. The method according to claim 1 , wherein each cycle of the ligase reaction is from about 30 seconds to about five minutes long.

8. The method according to claim 1 , wherein said subjecting the ligase reaction mixture to one or more ligase reaction cycles is repeated for 2 to 50 cycles.

9. The method according to claim 1 , wherein total time for said subjecting the ligase reaction mixture to one or more ligase reaction cycles is 1 to 250 minutes.

10. The 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, and Pyrococcus ligase.

11. The method according to claim 1 , wherein the target-specific portions of the oligonucleotide probes each have a hybridization temperature of 50-85° C.

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

13. The method according to claim 1 , wherein the oligonucleotide probe sets are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof.

14. 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:

producing one or more ligation products from a reaction mixture, wherein said reaction mixture comprises:

a ligase;

one or more target nucleotide sequences; and

one or more oligonucleotide probe sets, each said probe set including (a) a first oligonucleotide probe comprising a first target-specific portion capable of hybridizing to a corresponding target nucleotide sequence, an upstream primer-specific portion, and a further portion; and (b) a second oligonucleotide probe comprising a second target-specific portion capable of hybridizing to said corresponding target nucleotide sequence and a downstream primer-specific portion, wherein a ligation product comprising the first and the second target-specific portions is capable of being produced after the first and the second target-specific portions are hybridized to said corresponding target nucleotide sequence, but is not produced when the first and the second target-specific portions are hybridized with one or more mismatches to a nucleotide sequence present in said reaction mixture, wherein each of said one or more ligation products comprises a ligated sequence which includes (1) the upstream primer-specific portion, (2) the further portion, (3) first and second target-specific portions and (4) the downstream-primer specific portion, or complements thereof;

providing one or a plurality of oligonucleotide primer sets, each set comprising (a) an upstream primer and (b) a downstream primer;

subjecting said one or more ligation products to one or more polymerase chain reaction cycles to produce one or more extension products comprising the ligation product sequences and/or complements thereof; and

detecting the extension products to identify one or more target nucleotide sequences in the sample.

15. 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:

producing one or more ligation products from a reaction mixture, wherein said reaction mixture comprises:

a ligase;

one or more target nucleotide sequences; and

one or more oligonucleotide probe sets, each said probe set including (a) a first oligonucleotide probe comprising a first target-specific portion capable of hybridizing to a corresponding target nucleotide sequence, an upstream primer-specific portion, and a further portion; and (b) a second oligonucleotide probe comprising a second target-specific portion capable of hybridizing to said corresponding target nucleotide sequence and a downstream primer-specific portion, wherein a ligation product comprising the first and the second target-specific portions is capable of being produced after the first and the second target-specific portions are hybridized to said corresponding target nucleotide sequence, but is not produced when the first and the second target-specific portions are hybridized with one or more mismatches to a nucleotide sequence present in said reaction mixture, wherein each of said one or more ligation products comprises a ligated sequence which includes (1) the upstream primer-specific portion, (2) the further portion, (3) first and second target-specific portions and (4) the downstream-primer specific portion, or complements thereof;

providing one or a plurality of oligonucleotide primer sets, each set comprising (a) an upstream primer and (b) a downstream primer;

subjecting said one or more ligation products to one or more polymerase chain reaction cycles to produce one or more extension products, each extension product comprising the ligated sequence of a corresponding ligation product from which said extension product is amplified, wherein the further portion of the ligated sequence distinguishes said extension product from other extension products that comprise other ligated sequences or complements thereof;

capturing said one or more extension products to a solid support; and

detecting the identities of the further portions in said captured extension products to indicate the presence of one or more target nucleotide sequences in said reaction mixture.

Assignments (3)

Continuity (8)