Composition and method for the detection of the presence of a polynucleotide sequence of interest

• US 4,755,458 A
• Filed: 08/30/1984
• Issued: 07/05/1988
• Est. Priority Date: 08/30/1984
• Status: Expired due to Term

• Current Assignee: Enzo Diagnostics, Inc.
• Original Assignee: Enzo Biochem Incorporated
• Inventors: Rabbani, Elazar, Engelhardt, Dean L.
• Primary Examiner(s): Nucker, Christine M.
• Application Number:
  US06/646,171
• Time in Patent Office: 1,405 Days
• Field of Search: 435/6, 435/5, 435/29, 435/35, 435/39, 436/501, 436/508, 436/63, 436/94, 536/27, 536/28, 536/29, 935/77, 935/78, 935/81
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to a wide range of genetic analyses using the technique of nucleic acid hybridization. These genetic analyses include, for example, the diagnosis of infections by foreign microbes and the detection of specific genetic traits and abnormalities. More specifically, the present invention is related to the detection of the presence of a polynucleotide sequence of interest.

Claims

1. In a composition for detecting the presence of a polynucleotide sequence of interest in a sample of polynucleotide sequences comprising:

a composition comprising at least one first and at least one second polynucleotide the improvements comprising;

(a) said first and second sequences are labeled with a first detectable marker and are either present as separate molecules from which said first polynucleotide sequence has not been isolated or are covalently linked and further characterized in that said first sequence is capable of hybridizing to said sequence of interest and said second sequence is not capable of hybridizing to said sequence of interest and is not capable of hybridizing to said first sequence; and

(b) a composition comprising at least one third polynucleotide sequence that is unlabeled or is labeled with a second detectable marker and characterized in that said third sequence is not capable of hybridizing to said first sequence and is not capable of hybridizing to said sequence of interest, and further characterized in that said third sequence is;

(i) capable of hybridizing to said second sequence, such that by hybridizing to said second sequence said third sequence blocks hybridization between said second sequence and complementary non-target sequences that may be contained in the sample and decreased the likelihood that said second sequence will generate a false positive signal upon detection of said first detectable marker;

or

(b) substantially identical to said second sequence, such that by means of hybridizing to said complementary non-target sequences that may be contained in the sample said third sequence blocks hybridization between said second sequence and said complementary non-target sequences and decreases the likelihood that said second sequence will generate a false positive signal upon detection of said first detectable marker.

2. A composition as in claim 1 wherein said second polynucleotide sequence comprises a vector polynucleotide sequence.

3. A composition as in claim 1, wherein said first polynucleotide sequence is covalently linked to said second polynucleotide sequence in a chromosome.

4. A composition as in claim 3, wherein said second polynucleotide sequence further comprises a vector polynucleotide sequence.

5. A composition as in claims 3 or 4, wherein said first polynucleotide sequence is capable of hybridizing to a polynucleotide sequence selected from the group consisting of N. gonorrhoea, herpes simplex virus I, herpes simplex virus II, Brucella abortus, Bordetella pertussis, Shigella dysenteria, Haemophilus influenzae, Mycobacterium tuberculosis, Pseudomonas pseudomallei, Salmonella typhi, Salmonella typhimurium and N. meningitidis.

6. A composition as in claim 1, wherein said second polynucleotide sequence is a host polynucleotide sequence.

7. A composition as in claim 1, wherein said third polynucleotide sequence is unlabeled.

8. A composition as in claim 1, wherein said third polynucleotide sequence is present in an amount by weight from about 100 to about 1000 fold greater than said second polynucleotide sequence of said composition.

9. A composition as in claim 1, wherein said third polynucleotide sequence is from about 50 to about 250 nucleotides in length.

10. A composition as in claim 1 wherein said first and second detectable markers are selected from the group consisting of radioactive nuclides and chemical labels.

11. A composition as in claim 10, wherein said chemical label is selected from the group consisting of biotinated moieties, antigens, sugars, fluors, phosphors, enzymes, apoenzymes and cofactors, ligands, allosteric effectors, ferritin, dyes and microspheres.

12. In a method for the detection of a polynucleotide sequence of interest in a sample of polynucleotide sequences comprising:

contacting under hybridizing conditions said sample of polynucleotide sequences with a composition comprising at least one first and at least one second polynucleotide and detecting said polynucleotide of interest by means of said first detectable marker, the improvements comprising;

(a) said first and second sequences are labeled with a first detectable marker and are either present as separate molecules from which said first polynucleotide sequence has not been isolated or are covalently linked and further characterized in that said first sequence is capable of hybridizing to said sequence of interest and said second sequence is not capable of hybridizing to said sequence of interest and is not capable of hybridizing to said first sequence; and

(b) said sample is further contacted, prior to detecting, with a composition comprising at least one-third polynucleotide sequence that is unlabeled or is labeled with a second detectable marker and characterized in that said third sequence is not capable of hybridizing to said first sequence and is not capable of hybridizing to said sequence of interest, and further characterized in that said third sequence is;

(i) capable of hybridizing to said second sequence, such that by hybridizing to said second sequence said third sequence blocks hybridization between said second sequence and complementary non-target sequences that may be contained in the sample and decreases the likelihood that said second sequence will generate a false positive signal upon detection of said first detectable marker;

or

(ii) substantially identical to said second sequence, such that by means of hybridizing to said complementary non-target sequences that may be contained in the sample said third sequence blocks hybridization between said second sequence and said complementary non-target sequences and decreases the likelihood that said second sequence will generate a false positive signal upon detection of said first detectable marker.

13. A method in accordance with claim 12, wherein said first polynucleotide sequence is covalently linked to said second polynucleotide sequence in a chromosome.

14. A method in accordance with claim 13, wherein said first polynucleotide sequence is specific for a polynucleotide sequence selected from the group consisting of N. gonorrhoea, herpes simplex virus I, herpes simplex virus II, Brucella abortus, Bordetella pertussis, Shigella dysenteria, Haemophilus influenzae, Mycobacterium tuberculosis, Pseudomonas pseudomallei, Salmonella typhi, Salmonella typhimurium and N. meningitidis.

15. A method in accordance with claim 12, wherein said third polynucleotide sequence is from about 50 to about 250 nucleotides in length.

16. A method in accordance with claim 12 wherein said second polynucleotide sequence comprises a vector polynucleotide sequence.

17. A method in accordance with claim 12, wherein said second polynucleotide sequence is a host polynucleotide sequence.

18. A method in accordance with claim 12, wherein said third polynucleotide sequence is present in an amount by weight from about 100 to about 1000 fold greater than said second polynucleotide sequence.

19. A method according to claim 12, wherein said sample is derived from human nucleic acid material.

20. A method according to claim 12, comprising contacting under hybridizing conditions said composition of first and second sequences with at least one of said third sequence, wherein said third sequence is present in an amount by weight that is at least 100 fold greater than the amount by weight of said second sequence and, at about the same time, contacting under hybridizing conditions said composition of first and second sequences with said sample.

21. A method according to claim 12, comprising first contacting under hybridizing conditions said composition of first and second sequences with at least one of said third sequence characterized in that said third sequence is capable of hybridizing to said second sequence, wherein said third sequence is present in an amount by weight that is at least 100 fold greater than the amount by weight of said second sequence, and then, in a second step, contacting under hybridizing conditions said composition of first and second sequences with said sample.

22. A method according to claim 12, comprising first contacting under hybridizing conditions said sample with at least one of said third sequence characterized in that said third sequence is substantially identical to said second sequence, wherein said third sequence is present in an amount by weight that is at least 10 fold greater than the amount by weight of said complementary non-target sequences in the sample, and then, in a second step, contacting under hybridizing conditions said composition of first and second sequences with said sample.