DETECTION OF NUCLEIC ACID SEQUENCE DIFFERENCES USING COUPLED LIGASE DETECTION AND POLYMERASE CHAIN REACTIONS

• US 20080171330A1
• Filed: 10/31/2007
• Published: 07/17/2008
• Est. Priority Date: 05/29/1996
• Status: Active Grant

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University), Board of Supervisors of Louisiana State University Agricultural & Mechanical College (Louisiana State University System), Regents of The University of Minnesota (University of Minnesota)
• Inventors: BARANY, George, BARANY, Francis, HAMMER, Robert P., LUBIN, Matthew
• Granted Patent: US 7,556,924 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/131 : Allele specific probes
• CPC Class Codes: C12Q 2537/143 : Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/114 : involving a quantitation step
• CPC Class Codes: C12Q 2561/125 : Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays

Abstract

The present invention relates to a method for identifying a target nucleotide sequence. This method involves forming a ligation product on a target nucleotide sequence in a ligase detection reaction mixture, amplifying the ligation product to form an amplified ligation product in a polymerase chain reaction (PCR) mixture, detecting the amplified ligation product, and identifying the target nucleotide sequence. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence differences.

Claims

1-9. -9. (canceled)

10. 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 padlock oligonucleotide probes, each probe comprising (a) a first target-specific portion and a 5′

upstream primer-specific portion and (b) a second target-specific portion and a 3′

downstream primer-specific portion, wherein the first and second target-specific portions of a particular probe are suitable for ligation together when hybridized on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when first and second target-specific portions of a particular probe are hybridized to any other nucleotide sequence present in the sample;

providing a ligase;

blending the sample, the one or more padlock oligonucleotide probes, and the ligase to form a ligase detection reaction mixture;

subjecting the ligase detection reaction mixture to one or more ligase detection reaction cycles to form a ligation product sequence comprising (a) the 5′

upstream primer specific portion, (b) the target-specific portions, and (c) the 3′

downstream primer-specific portion, when the respective target nucleotide sequence of the corresponding padlock oligonucleotide probe 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 detection 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.

11. The method according to claim 10, 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.

12. The method according to claim 10, wherein the ligation product sequence of each padlock oligonucleotide probe 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.

13. The method according to claim 10, wherein each padlock oligonucleotide probe is configured so that the ligation product nucleotide sequence from each oligonucleotide probe 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 unique ligation product nucleotide sequences 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 unique nucleotide sequence portions to identify one or more target nucleotide sequences in the sample.

14. The method according to claim 10, wherein, in each primer set, one primer comprises a detectable reporter label and the other primer comprises an addressable array-specific portion which is linked to the 5′

end of that primer and remains single stranded after said subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles, 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 addressable array-specific portions 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 at particular sites to identify one or more target nucleotide sequences in the sample.

15. The method according to claim 10, 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 padlock oligonucleotide probes of a particular probe group, the padlock oligonucleotide probes forming a plurality of oligonucleotide probe groups, each group comprised of two or more padlock oligonucleotide probes, wherein padlock oligonucleotide probes 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.

16. The method according to claim 15, wherein padlock oligonucleotide probes 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 each padlock oligonucleotide probe 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.

17. The method according to claim 15, wherein the ligation product sequences of the padlock oligonucleotide probes comprise unique sequences so that they 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 unique nucleotide sequences of a given padlock oligonucleotide probe;

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; and

detecting the presence of extension products captured at particular sites.

18. The method according to claim 10, wherein each cycle of the ligase detection reaction is from about 30 seconds to about five minutes long.

19. The method according to claim 10, wherein said subjecting the ligase detection reaction mixture to one or more ligase detection reaction cycles is repeated for 2 to 50 cycles.

20. The method according to claim 10, wherein total time for said subjecting the ligase detection reaction mixture to one or more ligase detection reaction cycles is 1 to 250 minutes.

21. The method according to claim 10, wherein the ligase is selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, and Pyrococcus ligase.

22. The method according to claim 10, wherein the target-specific portions of the oligonucleotide probes each have a hybridization temperature of 50-85°

C.

23. The method according to claim 10, wherein the target-specific portions of the oligonucleotide probes are 20 to 28 nucleotides long.

24. The method according to claim 10, wherein the padlock oligonucleotide probes are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof.

25. 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 padlock oligonucleotide probes, each said probe including (a) a first target-specific portion capable of hybridizing to a corresponding target nucleotide sequence and (b) a second target-specific portion capable of hybridizing to said corresponding target nucleotide sequence, 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 first target-specific portion of the padlock oligonucleotide probe and (2) the second target-specific portion of the padlock oligonucleotide probe, or complements thereof,

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.

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:

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 padlock oligonucleotide probes, each said probe including (a) a first target-specific portion capable of hybridizing to a corresponding target nucleotide sequence and (b) a second target-specific portion capable of hybridizing to said corresponding target nucleotide sequence, 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 first target-specific portion of the padlock oligonucleotide probe and (2) the second target-specific portion of the padlock oligonucleotide probe, or complements thereof;

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 (1) the ligated sequence of a corresponding ligation product from which said extension product is amplified and (2) an addressable array-specific portion which 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 addressable array-specific portions in said captured extension products to indicate the presence of one or more target nucleotide sequences in said reaction mixture.