Gel microdroplets in genetic analysis

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.

What is claimed is:  
     
         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; 
 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;    isolating or detecting the at least one gel microdrop.    
     
     
         2 . The method of  claim 1 , wherein the biological entities are selected from the group consisting of cells, viruses, nuclei and chromosomes.  
     
     
         3 . The method of  claim 1 , wherein the biological entities are not fixed chemically before the contacting step.  
     
     
         4 . The method of  claim 1 , further comprising amplifying the nucleic acids before the contacting step.  
     
     
         5 . The method of  claim 1 , wherein the biological entities are chromosomes.  
     
     
         6 . The method of  claim 1 , 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.  
     
     
         7 . The method of  claim 1 , wherein the populations of gel microdrops is formed by forming a preparation of biologicial entities in a liquid gel and passing the preparation through a puslating orifice.  
     
     
         8 . The method of  claim 7 , wherein the pulsating orifice is a component of an ink jet printer.  
     
     
         9 . The method of  claim 1 , wherein most drops contain zero chromosomes, and 1-30% of drops contain a single chromosome.  
     
     
         10 . The method of  claim 1 , wherein the drops are 2-200 μm in diameter.  
     
     
         11 . The method of  claim 1 , wherein the gel is selected from agarose, alginate, carrageenan, or polyacrylamide.  
     
     
         12 . The method of  claim 1 , further comprising digesting the gel at least one isolated microdrop with agarase to isolate a nucleic acid within the microdrop.  
     
     
         13 . The method of  claim 1 , further comprising denaturing the nucleic acid in the microdrops before the contacting step.  
     
     
         14 . The method of  claim 1 , wherein the gel is agarose and the method further comprising crosslinking hydroxyl groups in the agarose with each other and with hydroxyl groups in the nucleic acid between the denaturation and contacting steps.  
     
     
         15 . The method of  claim 14 , wherein the hybridization is performed at a temperature of over 68° C. or in the presence of a formamide concentration greater than 20%.  
     
     
         16 . The method of  claim 5 , wherein the biological entities are are obtained from a human, nonhuman mammal, plant, bacterium, fungus, fish, or insect.  
     
     
         17 . The method of  claim 1 , wherein the probe is labelled.  
     
     
         18 . The method of  claim 17 , wherein the microdrops further comprise a reagent that amplifies a signal from the labelled probe.  
     
     
         19 . The method of  claim 17 , wherein the probe is labelled with an enzyme, and the reagent is a substrate for the enzyme.  
     
     
         20 . The method of  claim 17 , wherein the probe is fluorescently labelled.  
     
     
         21 . The method of  claim 1 , wherein the probe specifically hybridizes to a subpopulation of the microdrops each containing a nucleic acid bearing a complementary sequence to the probe.  
     
     
         22 . The method of  claim 1 , wherein the at least one gel microdrop is isolated by fluorescent activated cell sorting.  
     
     
         23 . The method of  claim 1 , wherein the at least one gel microdrop is detected by flow cytometry, microscopy, digital image analyzing, scanning cytometry, photon counting or ccd.  
     
     
         24 . The method of  claim 1 , wherein the probe is a nucleic acid.  
     
     
         25 . The method of  claim 24 , wherein the probe is a locus-specific probe.  
     
     
         26 . The method of  claim 5 , wherein the probe comprises first and second probes respectively complementary to different chromosomes in a wildtype individual, whereby co-hybridization of the first and second probes to the same chromosome indicates a chromosomal translocation in an individual.  
     
     
         27 . The method of  claim 1 , wherein the probe hybridizes to a satellite DNA sequence, centromeric, a telomeric region or a repetitive sequence.  
     
     
         28 . The method of  claim 1 , wherein the probe is a chromosome specific probe.  
     
     
         29 . The method of  claim 5 , wherein the population of chromosomes are obtained from a single cell or a homogeneous cell line from a patient.  
     
     
         30 . The method of  claim 1 , further comprising labelling microdrops containing an entity with a second label without labelling empty microdrops with the second label.  
     
     
         31 . The method of  claim 5 , wherein the population of chromosomes is obtained from a population of different cells in a patient.  
     
     
         32 . The method of  claim 31 , further comprising determining the ratio of a subpopulation of microdrops containing a chromosome hybridized to the probe to a supopulation of microdrops containing a chromosome not hybridized to the probe.  
     
     
         33 . The method of  claim 32 , wherein the ratio is less than 1:10.  
     
     
         34 . The method of  claim 32 , wherein the probe hybridizes to a nucleic acid segment bearing a mutation and the ratio indicates the proportion of cells in the population bearing the mutation.  
     
     
         35 . The method of  claim 32 , wherein the mutation is a somatic mutation.  
     
     
         36 . The method of  claim 32 , wherein the mutation is a germline mutation.  
     
     
         37 . The method of  claim 1 , further comprising contacting an isolated gel drop containing a nucleic acid with a restriction enzyme, whereby the restriction enzyme cleaves the nucleic acid within the drop.  
     
     
         38 . The method of  claim 5 , further comprising preparing a single chromosomal fragment library from a chromosome in an isolated gel microdrop.  
     
     
         39 . The method of  claim 5 , further comprising preparing probes from a single chromosome in an isolated gel microdrop.  
     
     
         40 . The method of  claim 39 , wherein the probes are chromosome painting probes.  
     
     
         41 . The method of  claim 39 , wherein the probes are reverse chromosome painting probes.  
     
     
         42 . The method of  claim 1 , further comprising storing a gel microdrop encapsulating a biological entity for at least one hour.  
     
     
         43 . The method of  claim 42 , wherein the biological entity is stored before the contacting step.  
     
     
         44 . The method of  claim 42 , wherein the biological entity is stored after the isolating or detecting step.  
     
     
         45 . The method of  claim 42 , wherein the gel microdrop is stored for at least six months.  
     
     
         46 . The method of  claim 5 , further comprising viewing the isolated gel microdrop under a microscope to determine which regions of the chromosome have hybridized to the probe.  
     
     
         47 . The method of  claim 1 , further comprising contacting the at least one gel drop containing a nucleic acid hybridized to the probe with a label that binds to the probe.  
     
     
         48 . The method of  claim 1 , wherein the population of chromosomes comprises at least 10,000 chromosomes.  
     
     
         49 . The method of  claim 1 , wherein the biological entities are cells and the probe hybridizes to an RNA molecule with the cells.  
     
     
         50 . 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;    contacting the microdrops with a first probe that is complementary to a nucleic acid segment containing the 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 the 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.    
     
     
         51 . The method of  claim 50 , wherein the disease is cancer.  
     
     
         52 . The method of  claim 51 , wherein the mutation occurs in a p53, BRCA-1, BRCA-2, ras or retinoblastoma gene.  
     
     
         53 . A method of chromosome analysis, comprising forming a population of gel micropdrops encapsulating a population of nucleic, whereby at least some microdrops in the population each encapsulate a single nucleus; 
 contacting the population of gel microdrops with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in at least one chromosome in a nucleus of least one gel microdroplet;    isolating or detecting the at least one gel microdroplet.    
     
     
         54 . A method of isolating chromosomes comprising: 
 culturing a population of cells in genistein and colcemid to synchronize chromosomes in metaphase;    isolating chromosomes from the cells.    
     
     
         55 . A method of isolating chromosomes comprising: 
 lysing a population of cells to form a lysate;    treating the lysate with an antibody linked to a magnetic particles, wherein the antibody specifically binds to one or more chromosomes in the cells;    isolating magnetic particles from the lysate.    
     
     
         56 . A method of chromosome analysis, comprising forming a population of gel micropdrops encapsulating a population of cells or nuclei, whereby at least some microdrops in the population each encapsulate a single nucleus; 
 contacting the population of gel microdrops with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in at least one nucleus in at least one gel microdrop;    isolating or detecting the at least one gel microdrop.    
     
     
         57 . The method of  claim 56 , wherein the probe is labelled with an enzyme and the gel microdrops contain a substrate for the enzyme.  
     
     
         58 . The method of  claim 56 , wherein gel microdrops are formed from a biotinylated gel, and the. substrate is linked to biotin via an avidin or streptavidin moiety.  
     
     
         59 . The method of  claim 58 , wherein the substrate is chemieluminescent.  
     
     
         60 . The method of  claim 59 , wherein the enzyme is horseradish peroxidase or alkaline phosphatase.  
     
     
         61 . The method of  claim 56 , wherein the detecting comprising analyzing the at least one gel microdrop with a fluorescence microscope, a digital image analyser, a scanning cytometer, a photon counting device or a ccd.  
     
     
         62 . The method of  claim 61 , wherein the detecting indicates the distribution of the probe within the nucleus of the at least one microdrop.  
     
     
         63 . The method of  claim 56 , wherein the detecting comprising placing a plurality of gel microdrops on a microscope slip and detecting a hybridization signal using a fluorescence microscope, a digital image analyser, a scanning cytometer, a photon counting device or a ccd.  
     
     
         64 . The method of  claim 56 , wherein the probe hybridizes to a single copy genomic sequence shorter than 50 kb.  
     
     
         65 . The method of  claim 56 , wherein the probe hybridizes to a single copy genomic sequence shorter than 10 kb.  
     
     
         66 . The method of  claim 56 , wherein the isolating or detecting is effected by flow cytometry, optionally with FACS, or MACS.  
     
     
         67 . A kit comprising a high melting temperature agarose, emulsification equipment, a label indicating how to use the kit for probe hybridization analysis.  
     
     
         68 . The kit of  claim 67 , further comprising at least one probe that hybridizes to a nucleic acid.