Detection of specific DNA sequences by flow cytometry

• US 4,770,992 A
• Filed: 11/27/1985
• Issued: 09/13/1988
• Est. Priority Date: 11/27/1985
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Barbara J. Trask, Van Den ENGH GER
• Inventors: Van den Engh, Gerrit J., Trask, Barbara J.
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/802,802
• Time in Patent Office: 1,021 Days
• Field of Search: 435/6, 935/78, 436/800, 436/805
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property

Abstract

A method for detection of DNA sequences. In the method, chromatin (comprising protein and DNA) is contacted with a cross-linking agent for the protein of the chromatin to provide a substantially rigid chromatin particle. The DNA of the chromatin particle is then subjected to treatment to cause a separation of the individual DNA strands into single stranded DNA. Preselected sequences of the single stranded DNA are then contacted with a complementary polynucleotide probe specific for the DNA sequence of interest. The polynucleotide probe is marked with a fluorescent label thereby labelling a target sequence of DNA in the chromatin particles. The fluorescently tagged DNA sequences are then detected by subjecting the polynucleotide probe to a suitable light source and detecting the light emitted by the fluorescent label so as to identify the preselected DNA sequence.

Claims

1. A method of detecting preselected DNA sequences in a chromatin particle by fluorescence activated flow cytometry or fluorescence microscopy comprising

(1) contacting chromatin with a cross-linking agent for the protein of said chromatin to provide a substantially rigid chromatin particle;

(2) subjecting the DNA of said chromatin particle to denaturing conditions so as to separate double stranded DNA into single stranded DNA;

(3) contacting preselected sequences of said single stranded DNA with a nucleic acid probe complementary to said preselected DNA sequence;

(4) marking said nucleic acid probe with a fluorescent label; and

(5) detecting said marked nucleic acid probe by fluorescence activated flow cytometry or fluorescence microscopy so to identify said preselected DNA sequence.

2. A method in accordance with claim 1 wherein said detection is accomplished by passing said chromatin particle through a fluorescence activated flow cytometer.

3. A method in accordance with claim 1 wherein said detection is accomplished by means of a fluorescence microscope.

4. A method in accordance with claim 1 wherein said cross-linking agent is an organic compound having at least two functional groups which are reactive with the terminal functional groups of the polar amino acids of said protein of said chromatin.

5. A method in accordance with claim 1 wherein said cross-linking agent is a diimidate having the formula:

##STR2## wherein R₁ and R₂ are selected from methyl, ethyl and propyl and n is an integer from 1 to 10.

6. A method in accordance with claim 5 wherein said cross-linking agent is dimethyl suberimidate.

7. A method in accordance with claim 1 wherein said chromatin particle is combined with an erythrocyte filler particle prior to said detection step.

8. A method in accordance with claim 7 wherein the ratio of said filler particles to said chromatin particles is from about 100:

1 to about 1000;

1.

9. A method in accordance with claim 7 wherein the ratio of said filler particles to chromatin particles is from about 300:

1 to about 700;

1.

10. A method in accordance with claim 7 wherein said filler particle has been contacted with a cross-linking agent for the protein of said erythrocyte.

11. A method in accordance with claim 10 wherein said cross-linking agent is an organic compound having at least two functional groups which are reactive with the terminal functional groups of the polar amino acids of said protein.

12. A method in accordance with claim 10 wherein said cross linking agent is a diimidate having the formula:

##STR3## wherein R₁ and R₂ are selected from methyl, ethyl and propyl and n is an integer from 1 to 10.

13. A method in accordance with claim 10 wherein said cross-linking agent is dimethyl suberimidate.

14. A method in accordance with claim 1 wherein said nucleic acid probe is marked by labeling the nucleic acid probe with a hapten or ligand and thereafter incubating said hapten or ligand labeled probe with an anti-hapten or anti-ligand antibody coupled to a fluorescent label.

15. A method in accordance with claim 1 wherein said chromatin particle contains the total nuclear DNA.

16. A method in accordance with claim 1 wherein said chromatin particle is a chromosome.

17. A method of detecting preselected DNA sequences in a chromatin particle by fluorescence activated flow cytometry or fluorescence microscopy comprising:

(1) contacting chromatin with a cross-linking agent for the protein of said chromatin to provide a substantially rigid chromatin particle,

(2) subjecting the DNA of said chromatin particle to denaturing conditions so as to separate double stranded DNA into single stranded DNA;

(3) contacting preselected sequences of said single stranded DNA with a nucleic acid probe complementary to said preselected DNA sequence, said nucleic acid probe being conjugated to a fluorescent label;

(4) detecting said fluorescently labeled nucleic acid probe by fluorescence activated flow cytometry or fluorescence microscopy so to identify said preselected DNA sequence.