Gel microdrops in genetic analysis

• US 6,586,176 B1
• Filed: 08/06/1999
• Issued: 07/01/2003
• Est. Priority Date: 08/07/1998
• Status: Expired due to Fees

• Current Assignee: Cellay, LLC
• Original Assignee: Cellay, LLC
• Inventors: McGrath, Patricia, Trnovsky, Jan
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US09/369,640
• Time in Patent Office: 1,425 Days
• Field of Search: 435/6, 435/91.2, 435/126, 435/325, 435/7.1, 536/123.1, 536/24.33, 536/24.3, 536/23.1, 536/120
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• 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: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2563/161 Vesicles, e.g. liposome
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The invention provides methods of nucleic acid analysis. Such methods entail forming a population of gel microdrops encapsulating a population of biological entities, each entity comprising a nucleic acid, whereby at least some microdrops in the population each encapsulate a single entity. The population of gel microdrops is then contacted with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in the nucleic acid in at least one gel microdrop. At least one gel microdrop is then analyzed or detected. The biological entities can be cells, viruses, nuclei and chromosomes.

Claims

1. A method of nucleic acid analysis, comprising

forming a population of gel microdrops encapsulating a population of biological entities, each entity comprising a nucleic acid, whereby at least some microdrops in the population each encapsulate a single entity;

crosslinking hydroxyl groups in the agarose of the microdrops with each other and with hydroxyl groups in the nucleic acids encapsulated in the microdrops;

contacting the population of gel microdrops with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in the nucleic acid in at least one gel microdrop;

analyzing the nucleic acid encapsulated with the microdrop by isolating or detecting the at least one gel microdrop;

wherein the cross-linking is reversible without damage to the nucleic acids encapsulated in the microdrops.

4. The method of any one of claims 1-3, wherein the biological entities are selected from the group consisting of cells, viruses, nuclei and chromosomes.

5. The method of any one of claims 1-3, wherein the biological entities are not fixed chemically before the contacting step.

6. The method of any one of claims 1-3, wherein the biological entities are chromosomes.

7. The method of any one of claims 1-3, wherein the population of gel microdrops is formed by forming a preparation of biological entities in a liquid gel, and dispersing the preparation into a hydrophobic solvent to form drops encapsulating the entities.

9. The method of any one of claims 1-3, wherein most drops contain zero chromosomes, and 1-30% of drops contain a single chromosome.

10. The method of any one of claims 1-3, wherein the drops are 2-200 μ

m in diameter.

11. The method of any one of claims 1-3, wherein the gel is selected from agarose, alginate, carrageenan, or polyacrylamide.

12. The method of any one of claims 1-3, wherein the biological entities are obtained from a human, nonhuman mammal, plant, bacterium, fungus, fish, or insect.

13. The method of claim 6, wherein the population of chromosomes are obtained from a single cell or a homogeneous cell line from a patient.

14. The method of any one of claim 1-3, further comprising labelling microdrops containing an entity with a second label without labelling empty microdrops with the second label.

15. The method of claim 6, wherein the population of chromosomes is obtained from a population of different cells in a patient.

16. The method of any one of claims 1-3, further comprising storing a gel microdrop encapsulating a biological entity for at least one hour.

17. The method of claim 16, wherein the biological entity is stored before the contacting step.

19. The method of claim 16, wherein the gel microdrop is stored for at least six months.

2. A method of nucleic acid analysis, comprising

forming a population of gel microdrops encapsulating a population of biological entities, each entity comprising a nucleic acid, whereby at least some microdrops in the population each encapsulate a single entity;

crosslinking hydroxyl groups in the agarose of the microdrops with each other and with hydroxyl groups in the nucleic acids encapsulated in the microdrops;

contacting the population of gel microdrops with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in the nucleic acid in at least one gel microdrop;

analyzing the nucleic acid encapsulated with the microdrop by isolating or detecting the at least one gel microdrop; and

releasing the nucleic acids from the microdrops after the contacting step.

3. A method of nucleic acid analysis, comprising

forming a population of gel microdrops encapsulating a population of biological entities, each entity comprising a nucleic acid, whereby at least some microdrops in the population each encapsulate a single entity;

crosslinking hydroxyl groups in the agarose of the microdrops with each other and with hydroxyl groups in the nucleic acids encapsulated in the microdrops;

contacting the population of gel microdrops with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in the nucleic acid in at least one gel microdrop;

analyzing the nucleic acid encapsulated with the microdrop by isolating or detecting the at least one gel microdrop;

wherein the cross-linking is performed using divinyl sulfone.

8. A method of nucleic acid analysis, comprising

forming a population of gel microdrops encapsulating a population of biological entities, each entity comprising a nucleic acid, whereby at least some microdrops in the population each encapsulate a single entity;

crosslinking hydroxyl groups in the agarose of the microdrops with each other and with hydroxyl groups in the nucleic acids encapsulated in the microdrops;

contacting the population of gel microdrops with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in the nucleic acid in at least one gel microdrop;

analyzing the nucleic acid encapsulated with the microdrop by isolating or detecting the at least one gel microdrop;

wherein the populations of gel microdrops is formed by forming a preparation of biological entities in a liquid gel and passing the preparation through a pulsating orifice of an ink jet printer.

18. A method of nucleic acid analysis, comprising

forming a population of gel microdrops encapsulating a population of biological entities, each entity comprising a nucleic acid, whereby at least some microdrops in the population each encapsulate a single entity;

crosslinking hydroxyl groups in the agarose of the microdrops with each other and with hydroxyl groups in the nucleic acids encapsulated in the microdrops;

contacting the population of gel microdrops with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in the nucleic acid in at least one gel microdrop;

analyzing the nucleic acid encapsulated with the microdrop by isolating or detecting the at least one gel microdrop; and

storing a gel microdrop encapsulating a biological entity for at least one hour after the isolating or detecting step.

20. A method of diagnosing a disease due to a mutation, comprising:

obtaining a sample of cells from a patient;

encapsulating a population of chromosomes from the sample in a population of microdrops;

crosslinking hydroxyl groups in microdrops with each other and with hydroxyl groups in the nucleic acid between the denaturation and contacting steps

contacting the microdrops with a first probe that is complementary to a nucleic acid segment containing a somatic mutation, and a second probe complementary to the chromosome in which the somatic mutation occurs at a site distal to the somatic mutation, whereby the first probe hybridizes to microdrops bearing the chromosome with a somatic mutation and the second probe hybridizes to microdrops bearing the chromosome irrespective whether the somatic mutation is present;

determining a ratio of microdrops hybridizing to the first probe and hybridizing to the second probe;

diagnosing the existence or prognosis of the disease from the ratio.

21. The method of claim 20, wherein the disease is cancer.

22. The method of claim 20, wherein the mutation occurs in a p53, BRCA-1, BRCA-2, ras or retinoblastoma gene.