DNA detection system

• US 4,978,608 A
• Filed: 09/04/1987
• Issued: 12/18/1990
• Est. Priority Date: 09/04/1987
• Status: Expired due to Term

• Current Assignee: Molecular Devices
• Original Assignee: Molecular Devices
• Inventors: Kung, Viola T., Nagainis, Peter A.
• Primary Examiner(s): Nucker, Christine
• Assistant Examiner(s): Krupen, Karen I.
• Application Number:
  US07/093,361
• Time in Patent Office: 1,201 Days
• Field of Search: 435/6, 435/7, 935/77, 935/78, 436/94, 436/501
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Picogram amounts of DNA can be detected in a sample by the use of high affinity single-stranded DNA binding proteins. The assay is applicable not only to pure DNA samples but also to samples containing significant amounts of protein.

Claims

1. A method for quantitatively detecting DNA in a sample wherein any DNA to be detected is labelled with a detectable label, said method comprising:

contacting a solid support with a high affinity single-stranded DNA binding protein (BP) to form immobilized BP;

contacting any single-stranded DNA in said sample with said immobilized BP to form DNA-BP complexes;

detecting any DNA-BP complexes by means of said label wherein any DNA-BP complexes detected are indicative of the presence of DNA in said sample.

2. A method according to claim 1, wherein said sample is subjected to DNA denaturing conditions prior to said contacting.

9. A method according to any one of claims 1, 3, or 5, wherein said BP is single-stranded DNA binding protein, T⁴ gene 32 protein or topoisomerase I.

10. A method according to any one of claims 1, 3, or 5, wherein said label is an enzyme joined to said BP.

11. A method according to claim 10, wherein said enzyme is urease or horseradish peroxidase.

12. A method according any one of claims 1, 3, or 5, further comprising:

additionally treating at least one background solution containing no DNA and at least one reference solution containing a known amount of DNA according to the steps of any one of claims 1, 3, or 5;

subtracting the label bound in said background solution from any DNA-BP complexes detected for said sample and from the label detected in said reference solution to produce an adjusted signal for said sample and for said reference solution; and

relating the adjusted signals for said sample and said reference solution to determine the amount of DNA present in said sample.

13. A method according to any one of claims 1, 3, or 5, wherein said sample further comprises protein.

14. A method according to claim 13, wherein said protein-containing sample is deproteinized prior to said contacting.

15. A method according to claim 14, wherein said protein-containing sample is deproteinized by a method comprising:

admixing said protein-containing sample with (a) a suspension comprising glass particles and (b) sufficient iodide to cause any DNA in said protein-containing sample to bind to said glass particles;

isolating said glass particles; and

eluting any DNA from said particles.

16. A method according to claim 15, wherein said eluting is carried out with water or phosphate buffered saline.

17. A method according to claim 14, wherein said protein-containing sample is deproteinized by a method comprising:

admixing said protein-containing sample with a proteolytic anzyme composition under conditions whereby protein present is hydrolyzed.

18. A method according to claim 17, further comprising:

after said admixing, separating any product of said hydrolyzed protein from any DNA.

19. A method according to claim 18, wherein said separating comprises:

after said admixing, centrifuging said sample through a membrane with a low molecular weight cutoff or passing said sample through an ultrafiltration device with a low molecular weight cutoff; and

recovering any DNA present.

20. A method according to claim 17, wherein said proteolytic enzyme composition comprises at least one of pronase or proteinase K.

23. A method according to claims 1, 3, or 5, wherein the high affinity single-stranded DNA binding protein has an affinity for single-stranded DNA of at least about 10⁵ M^-1.

24. A method according to any one of claims 1, 3, or 5, wherein the high affinity single-stranded DNA binding protein has an affinity for single-stranded DNA of about 10⁸ to about 10¹⁰ M^-1.

25. A method according to claim 24, wherein the single-stranded DNA has at least 100 nucleotides.

26. A method according to any one of claims 1, 3, or 5, wherein the high affinity single-stranded DNA binding protein has a single-stranded DNA binding affinity such that the high affinity single-stranded DNA binding protein may be eluted from single-stranded DNA-cellulose or single-stranded DNA-Sepharose by a sodium chloride concentration of greater than about 0.4M.

27. A method according to any one of claims 1, 3, or 5, wherein the high affinity single-stranded DNA binding protein has a single-stranded DNA binding affinity such that the high affinity single-stranded DNA binding protein may be eluted from single-stranded DNA-cellulose or single-stranded DNA-Sepharose by a sodium chloride concentration of greater than about 0.6M.

3. A method for quantitatively detecting DNA in a sample using a high affinity single-stranded DNA binding protein (BP), wherein said BP is labelled with a detectable label, said method comprising:

denaturing any DNA present in said sample;

binding any denatured DNA non-diffusively to a solid support;

contacting said solid support with BP to form DNA-BP complexes;

freeing said solid support of any unbound sample and BP; and

detecting said complexes by means of said label wherein any DNA-BP complexes detected are indicative of the presence of DNA in said sample.

4. A method according to claim 3, wherein said solid support is a positively charged nylon membrane or a nitrocellulose membrane.

5. A method for quantitatively detecting DNA in a sample, said method comprising:

contacting a solid support with a high affinity single-stranded DNA binding protein (BP) to form immobilized BP;

admixing said sample with a photoreactive compound labelled with a detectable label to form a mixture;

irradiating said mixture with activating light to react said photoreactive compound with any DNA present in said sample;

contacting said mixture with said immobilized BP to form DNA-BP complexes;

freeing said solid support of any mixture not specifically bound; and

detecting any photoreactive compound present on said solid support by means of said label wherein any DNA-BP complexes detected are indicative of the presence of DNA in said sample.

6. A method according to claim 5, wherein said photoreactive compound is isopsoralen.

7. A method according to claim 6, wherein said detectable label is biotin.

8. A method according to claim 7, wherein said biotin is detected using a conjugate comprising avidin, streptavidin or antibiotin joined to an enzyme, radioisotope or fluorophore.

21. A method according to claim 5, wherein said detectable label is a radionuclide or biotin.

22. A method according to claim 5, wherein said mixture is subjected to DNA denaturing conditions prior to said contacting.