Detection of specific sequences in nucleic acids

• US 5,962,223 A
• Filed: 12/19/1995
• Issued: 10/05/1999
• Est. Priority Date: 12/13/1984
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Hunkapiller, Michael W., Glazer, Alexander N., Whiteley, Norman M.
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US08/574,826
• Time in Patent Office: 1,386 Days
• Field of Search: 435/6, 435/5, 435/810, 435/25.4, 435/91.5, 435/91.52, 536/24.3, 536/24.31, 536/24.33, 536/24.32, 935/6, 935/8, 935/78
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2521/501 Ligase
• 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 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The invention provides a method for diagnosis of genetic abnormalities or other genetic conditions which can be readily automated. The method is used to determine the presence or absence of a target sequence in a sample of denatured nucleic acid and entails hybridizing the sample with a probe complementary to a diagnostic portion of the target sequence (the diagnostic probe), and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe), under conditions wherein the diagnostic probe remains bound substantially only to the sample nucleic acid containing the target sequence. The diagnostic probe and contiguous probe are then covalently attached to yield a target probe which is complementary to the target sequence, and the probes which are not attached are removed. In the preferred mode, one of the probes is labeled so that the presence or absence of the target sequence can then be tested by melting the sample nucleic acid-target probe duplex, eluting the dissociated target probe, and testing for the label. In another embodiment, the testing is accomplished without first removing probes not covalently attached, by attaching a hook to the probe that is not labeled, so that the labeled target probe may be recovered by catching the hook. In both instances, the presence of both the diagnostic probe and the contiguous probe is required for the label to appear in the assay. The above method is then applied to the detection of genetic diseases.

Claims

1. A kit for determining the presence or absence of a target polynucleotide sequence in a nucleic acid sample, said kit comprising:

a first probe which is complementary to a first region of a target sequence,

a second probe which is complementary to a second region of the target sequence, where said first and second target regions are contiguous with one another, and said first and second probes are capable of being ligated to each other when hybridized to such contiguous first and second target regions,

a third probe which is complemnentary to a third target region of the target sequence which is continuous with the first target region, such that said second and third target regions correspond to alternative allelic sequences or to a normal and mutated sequence, and

ligation means capable of ligating first and second probes, or first and third probes, that have hybridized specifically to contiguous target regions to which the first and second probes, or first and third probes, are complementary, respectively.

2. The kit of claim 1, wherein said ligation means is a ligase enzyme.

3. The kit of claim 1, wherein at least one of said probes contains a label that allows specific detection of said labeled probe in the presence of unlabeled nucleic acids.

4. The kit of claim 3, wherein said label is radioactive.

5. The kit of claim 3, wherein said label is a fluorescent label.

6. The kit of claim 3, wherein said label is an enzyme.

7. The kit of claim 1, wherein at least one of said probes includes non-polynucleotide hook moiety for capture of the probe on a support.

8. The kit of claim 7, wherein said hook moiety is a biotin moiety.

9. The kit of claim 7, wherein said hook moiety is an antigen, and the kit further includes an antibody which is immunospecific for the antigen.

10. The kit of claim 7, wherein said hook moiety is a ligand, and the kit further includes a receptor which can bind specifically to the ligand.

11. The kit of claim 1, which further includes instructions for performing ligation of the first and second probes, or first and third probes, when bound to contiguous target regions, to detect whether a target polynucleotide sequence complementary to the first and second probes, or first and third probes, is present in the sample.

12. The kit of claim 11, wherein said ligation means is a ligase enzyme.

13. The kit of claim 11, wherein at least one of said probes contains a label that allows specific detection of said labeled probe in the presence of unlabeled nucleic acids.

14. The kit of claim 13, wherein said label is radioactive.

15. The kit of claim 13, wherein said label is a fluorescent label.

16. The kit of claim 13, wherein said label is an enzyme.

17. The kit of claim 11, wherein at least one of said probes includes a non-polynucleotide hook moiety for capture of the probe on a support.

18. The kit of claim 17, wherein said hook moiety is a biotin moiety.

19. The kit of claim 17, wherein said hook moiety is an antigen, and the kit further includes an antibody which is immunospecific for the antigen.

20. The kit of claim 17, wherein said hook moiety is a ligand, and the kit further includes a receptor which can bind specifically to the ligand.