Nucleic acid sequence detection employing probes comprising non-nucleosidic coumarin derivatives as polynucleotide-crosslinking agents

• US 6,277,570 B1
• Filed: 09/04/1998
• Issued: 08/21/2001
• Est. Priority Date: 04/13/1993
• Status: Expired due to Fees

• Current Assignee: Douglas Y. Thien
• Original Assignee: Naxcor
• Inventors: Van Atta, Reuel B., Albagli, David, Cheng, Peter C., Huan, Bingfang, Wood, Michael L., Thien, Douglas Y.
• Primary Examiner(s): Gitomer, Ralph
• Assistant Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US09/149,161
• Time in Patent Office: 1,082 Days
• Field of Search: 435/6, 536/24.3, 536/24.31, 536/24.32
• US Class Current: 435/6.11
• CPC Class Codes: B01L 2300/1883 using thermal insulation
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C07D 311/16 substituted in position 7
• CPC Class Codes: C07D 407/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07F 9/65522 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Methods and compositions are provided for detecting nucleic acid sequences. Probes comprising a crosslinking agent are combined with a sample which may comprise a target sequence which is complementary to the probe. Hybridization is allowed to occur between complementary sequences. The crosslinking agent is activated. Covalent bonds are formed between the probe and the target sequence if they are hybridized to one another. The crosslinked nucleic acids can then be detected to indicate the presence of the target sequence. Also provided are kits comprising reagents.

Claims

1. A method of detecting a target nucleic acid in a sample, said method comprising the steps of:

a) hybridizing a target nucleic acid to a capture probe, said capture probe characterized bv having;

i) a polynucleotide having a sequence homologous to that of said target nucleic acid;

ii) a non-nucleosidic coumarin derivative incorporated into the backbone of said polynucleotide such that it replaces one or more nucleotides otherwise complementary to said target nucleic acid, wherein said coumarin derivative acts as a photoactivatable polynucleotide-crosslinking agent; and

iii) optionally at least one label;

b) activating said crosslinking agent, whereby a covalent crosslink occurs between said probe and said target nucleic acid; and

c) detecting the presence of the crosslinked nucleic acid pair as indicative of the presence of said target sequence in said sample.

2. The method of claim 1 wherein the target nucleic acid is from a microorganism selected from the group consisting of Chiamydia, Neisseria, Mycobacterium, Mycoplasma, Legionella and Listeria monocytogenes.

3. The method of claim 1 wherein the target nucleic acid is from a microorganism selected from the group consisting of Haemophilus ducreyi, Treponema pallidum, Helicobacter pylori, Pneumocystis carinii, Borrelia burgdorferi Salmonella, HIV I and II, HTLV-II, Hepatitis A, B, C, and D, Cytomegalovirus, human Papillomavirus, Respiratory syncytial virus, Epstein-Barr virus, Dengue virus, Eastern and Western Encephalitus viruses, Ebola virus, and Lassa virus.

4. The method of claim 1, wherein the target nucleic acid is selected from the group consisting ofthe TEM-1 gene (β

-lactamase) in Enterobactenaceae;

the TEM-1 gene in penicilinase producing N. gonorrhoeae (PPNG);

the gene conferring aminoglycoside antibiotic resistance;

the gene conferring erythromycin resistance;

the gene conferring rifampin resistance and the gene conferring vancomycin resistance.

5. The method of claim 1 where in the target nucleic acid is selected from the group consisting of fetal DNA, genes indicating sex of a fetus, the factor V gene, the factor V gene having the Leiden mutation, the hemochromatosis gene, the factor II gene and a gene known to be unique to human chromosome 21.

6. The method of claim 1 wherein said capture probe is characterized by having at least one label.

7. The method of claim 1 wherein said label is selected from the group consisting of an antibody ligand, a lectin-bindin sugar and biotin.

8. The method of claim 1, wherein said label is selected from the group consisting of a chemiluminescer, a radiolabel and a fluorophore.

9. The method of claim 1 further comprising the step of:

hybridizing at least one reporter probe to said target nucleic acid, said reporter probe being characterized by having;

i) a polynucleotide having a sequence that is complementary to that of said target nucleic acid; and

ii) a label, but not

iii) a photoactivatable polynucleotide-crosslinking agent.

10. The method of claim 9 wherein said label of said reporter probe is a fluorophore.

11. The method of claim 1 further comprising the step of:

d) isolating said crosslinked nucleic acid pair.

12. The method of claim 1 wherein said target nucleic acid in a sample is genomic DNA in whole blood.

13. A method according to claim 1, further comprising:

d) simultaneously performing said hybridization and said activation steps for one or more additional target nucleic acids, and/or simultaneously performing said hybridization, activation and detection steps in one or more additional samples in an automated system.

14. A kit including components for detecting a target nucleic acid in a sample, said kit comprising:

a) a capture probe characterized by having;

i) a polynucleotide having a sequence homologous to that of said target and capable of hybridizing with said target nucleic acid;

ii) a non-nucleosidic coumarin derivative incorporated into the backbone of said polynucleotide such that it replaces one or more nucleotides otherwise complementary to said target nucleic acid, wherein said coumarin derivative acts as a photoactivatable polynucleotide-crosslinking agent; and

iii) a label.

15. A kit according to claim 14 further comprising:

b) at least one reporter probe, said reporter probe characterized by having;

i) a polynucleotide having a sequence that is complementary to that of said target nucleic acid; and

ii) a label, but not

iii) a photoactivatable polvnucleotide-crosslinking agent.

16. The kit of claim 14 further comprising:

b) a chart of standardized results obtained using said kit with samples that are known to contain and samples that are known not to contain said target nucleic acid.

17. The kit of claim 14 further comprising:

a control probe, said control probe having;

i) a polynucleotide having a sequence homologous to that of a target nucleic acid; and

ii) a non-nucleosidic coumarin derivative incorporated into the backbone of said polynucleotide such that it replaces one or more nucleotides otherwise complementary to said target nucleic acid, wherein said coumarin derivative acts as a photoactivatable polynucleotide-crosslinking agent.

19. The kit of claim 14, wherein said label is selected from the group consisting of an antibody ligand, a lectin-binding sugar and biotin.

20. The kit of claim 14, wherein said label is selected from the group consisting of a chemiluminescer, a radiolabel and a fluorophore.

21. The kit of claim 14, further comprising a control sample, wherein said control contains a known amount of said target nucleic acid.

18. A method of confirming the detection of a target nucleic acid in a sample, said method comprising the steps of:

a) hybridizing said target nucleic acid in a first aliquot of said sample with a labeled probe, said labeled probe characterized by having;

i) a polynucleotide having a sequence homologous to that of said target nucleic acid;

ii) a non-nucleosidic coumarin derivative incorporated into the backbone of said polynucleotide such that it replaces one or more nucleotides otherwise complementary to said target nucleic acid, wherein said coumarin derivative acts as a photoactivatable polynucleotide-crosslinking agent; and

iii) alabel;

b) hybridizing said target nucleic acid in a second aliquot of said sample with a mixture of said labeled probe and unlabeled probe, said unlabeled probe characterized by having;

i) a polynucleotide having a sequence homologous to that of said target nucleic acid;

ii) a non-nucleosidic coumarin derivative incorporated into the backbone of said polynucleotide such that it replaces one or more nucleotides otherwise complementary to said target nucleic acid, wherein said coumarin derivative acts as a photoactivatable polynucleotide-crosslinking agent;

c) activating said crosslinking agent in each aliquot, whereby a covalent crosslink occurs between each of said probes and said target nucleic acid;

d) detecting the amount of labeled crosslinked nucleic acid pairs in each aliquot; and

e) comparing said amounts with the relative proportions of labeled and unlabeled probe in said first and second aliquots as an indication of the specificity of said detection.

22. The method of claim 18, wherein said label is selected from the group consisting of an antibody ligand, a lectin-binding sugar and biotin.

23. The method of claim 18, wherein said label is selected from the group consisting of a chemiluminescer, a radiolabel and a fluorophore.