Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6841—In situ hybridisation

Definitions

• the human genome consists of about 3 billion base pairs of DNA carried on 46 chromosomes. This genetic blueprint encodes the information required for the growth, differentiation, maintenance and proper functioning of human cells. To aid in identifying genes associated with disease . there has been great interest in mapping the human genome. Indeed, in creating the Human Genome Project, the United States government has announced the elucidation of the human genome as a national objective.
• the two main types of human genome maps are genetic linkage and physical. Genetic linkage maps are generated mainly by studying families and measuring the frequency with which two different traits are inherited together, or linked. Physical maps, on the other hand, are derived from measurements made on the DNA molecules that form the human genome.
• Physical maps can be created based on information provided by restriction fragment polymorphisms or a collection of ordered clones of genomic fragments (e.g. cosmids). Maps based on ordered clones are especially useful, since each fragment is available as a clone that can be propagated and distributed. These clones can serve as a starting point for gene isolation, functional analyses and for the determination of nucleotide sequences. Preparing an ordered-cione collection involves cloning DNA fragments, determining their order in the genome and propagating the fragments in pure form to make them widely available for subsequent analysis. Because of the limited resolution currently available, the most difficult aspect involves determining the order of DNA fragments.
• In situ hybridization analysis has been useful for genome mapping, because it allows direct detection of the location at which a DNA fragment maps on a particular chromosome.
• the resolution for localizing probes to chromosomal bands in mef ⁇ ph ⁇ se chromosomes is only several megabases (Lichter, P., et a]., et. al.. Science 247:64-69 (1990); Lawrence. J.B., et. al.. Science 249:64-69 (1990)).
• FISH has been applied to cell targets collected in interphase (Lawrence, J.B. et. al.. Cell 52:51-61 (1988)).
• the invention relates to methods for ordering at least two discrete DNA sequences on a chromosome.
• the invention features a) contacting a eukaryotic cell containing a chromosome of interest with an agent that effects decondensation; b) preparing the cell containing decondensed chromatin for hybridization; and c) hybridizing the cell produced in step b) with at least two DNA probes, each probe being complementary to at least one discrete DNA sequence present on the chromosome of interest; and d) detecting the presence and relative order of the probes as an indication of the relative and absolute locations of the DNA sequences on the chromosome.
• interphase chromatin By decondensing interphase chromatin according to the method of the invention, high resolution interphase mapping can be carried out. In effect, fragments containing DNA sequences that abut one another or even overlap can be resolved. In addition, because the methods disclosed herein render the DNA more accessible to hybridization, results are more readily obtained.
• the invention features ordering at least two discrete DNA sequences on a fragment of DNA that has been extended, for example by physical means, chemical means or a combination of both.
• a preferred physical means for extending DNA is by gentle smearing.
• a preferred chemical means for obtaining extended DNA from a eukaryotic genome is by contacting the chromatin with a solution that removes histones.
• the DNA can be prepared for hybridization and hybridized with at least two DNA probes, each probe being complementary to at least one discrete DNA sequence present on the chromosome of interest. Once hybridized, the presence and relative order of the probes can be determined as an indication of the relative and absolute locations of the DNA sequences on the fragment of DNA.
• the subject invention is based on the discovery that chromosomes which are "relaxed” or DNA which is “extended” can be analyzed using a hybridization assay to determine the order in which a set of DNA sequences (i.e. at least two) map on a particular chromosome or fragment of DNA.
• the invention therefore, features two convenient methods for ordering a set of discrete DNA sequences at a high resolution.
• the ordering of discrete DNA sequences is useful for example for physical mapping.
• the term "ordering" means establishing the linear relationship of discrete DNA sequences relative to one another and/or relative to a known marker on a chromosome or portion of a chromosome.
• a cell is contacted with an agent that effects decondensation.
• an agent that effects decondensation prepared for hybridization and analyzed using at least two DNA probes, each probe being complementary to at least one discrete DNA sequence present on a chromosome of interest.
• the cells are in interphase, so that the chromatin is already in a somewhat extended form.
• the growth of cells in culture can be synchronized using known methods. By using synchronized cells, a higher proportion of cells in the interphase stage of the cell cycle can be obtained.
• an agent that effects decondensation can be a chemical which inhibits proteins that deacetylate histones (i.e. the set of proteins that interact with DNA to form chromatin).
• histones are hyperacetylated, the chromatin becomes less condensed or relaxed.
• a salt of a short chain fatty acid such as sodium butyrate. sodium propionate and sodium valerate
• when added to cells appears to relax chromatin, apparently by inhibiting the proteins that deacetylate histones.
• Cells containing relaxed chromatin are then prepared for in situ hybridization analysis using methods, which are well-known in the art. In general such methods involve depositing cells on a solid substrate, fixing, drying and denaturing them to yield single stranded DNA that is therefore available for hybridization.
• Cells can be deposited on a substrate such as glass, plastic ornitrocellulose.
• a glass microscope slide is preferred, because it can be readily manipulated and viewed under a microscope.
• the substrate can be pretreated with a "cell adherent" which improves the likelihood that a cell settling onto the surface remains attached during subsequent manipulations.
• a preferred cell adherent is 3-Aminopropyltriethoxysilane. Treatment with this adherent results In "silanized" substrates.
• adherents include poly-L lysine and mussel adhesin.
• Pretreatment of solid substrates can be accomplished using any method that ensures that a cell adherent is deposited (e.g. submersion, transferring using a dropper, etc.). Pretreated solid substrates can be stored in a dust free environment at room temperature. Cells on a substrate can then be processed through a fixation protocol to preserve the nuclei/ chromosome in a morphologically stable state so that nucleic acids are retained through the rigorous conditions present during in situ hybridization. Appropriate fixatives are well-known in the art and include, for example, 4%
• PBS phosphate buffered solution
• a fixative containing 3 parts ethanol and 1 part acetic acid, Carnoy's fixative, 1% osmium tetraoxide, Bouin's fixative, Zenker's fixative.
• deoxyribonucleic acid (DNA) probes labelled with a detectable marker can be prepared from single-stranded DNA molecules or fragments thereof according to procedures which are well known in the art. Such techniques include incorporotion of radioactive labels, direct attachment of fluorochromes or enzymes, and various chemical modifications of the nucleic acid fragments that render them detectable immunochemically or by other affinity reactions.
• a preferred method of labelling is by nick translation using a haptenated nucleotide triphosphate (e.g. biotin labelled dUTP) or by random primer extension (Feinberg 8. Vogelstein, Anal. Biochem. 137:266-267 (1984) (e.g.
• FISH fluorescent in situ hybridization
• the invention features ordering at least two discrete DNA sequences on a fragment of DNA that has been extended.
• DNA can be obtained, for example from a eukaryotic or a prokaryotic chromosome, although histones must first be removed from DNA obtained from a eukaryotic chromosome. 5 Histones can be removed chemically using a histone removing solution (e.g. see Paulson and Laemmli Cell 12:817-828 (1977)).
• DNA free in solution e.g. DNA extracted from a prokaryote or DNA extracted from a eukaryotic chromosome and treated to remove proteins
• DNA free in solution can be extended by physical means.
• a preferred physical means for extending DNA is by gentle smearing 0 or pulling of the DNA. e.g. using a pippette. Once extended.
• DNA can be prepared for hybridization and hybridized with appropriate probes as described above to determine the relative and absolute locations of the DNA sequences on the fragment of DNA.
• Cells were resuspended in BT buffer (20mM sodium butyrate, 1 mM Tris.25mM CI.0.9mM MgCI 2 and 0.9mM CaCI 2 at pH 7.6).
• the cell suspension was dropped from a height of about 2 ft onto humid slides and allowed to dry slowly in a humid atmosphere.
• the culture media was removed by aspiration and 0.075M KCI with 0.01% Triton was added, and culture incubated for 20 m at 37°C.
• Example 3 Ordering Cosmnids from Contigs which Map to Human Chromosomes 18 and 21 to Demonstrate the Resolution Achieved by the Methods Described in Examples 1 and 2.
• Probes consisted of cosmids from a chromosome 18 or chromosome 21 contig.
• Chromosome 18 contig This contig maps to chromosome 18q23 and comprises five cosmids spanning 109 kb. This DNA molecule can be obtained from the American Type Culture Collection (Rockville, MD) under Accession Number 68934. The two flanking cosmids (pWE7 (labelled with a green fluorophore)) and pWE50wl (labelled with a green fluorophore) and the central cosmid (pWE50 (labelled with a red fluorophore)) were used in these experiments. There is known to be a large gap between pWE50 and pWE50wl; restriction digests indicate that pWE7 and pWE50 and may overlap.
• Chromosome 21 contig This contig maps to chromosome 21q22.3 and consists of five cosmids extending l l ⁇ kb. The two flanking cosmids (CHC1-8A and pWE).
• Hybridization cocktails contained 5-8 ng/uL of each flanking cosmid labeled with digoxigenin-dUTP of biotin and 5-8 ng/uL of the central cosmid labeled with the alternate hapten.200 ng/uL human Cot-1 DNA. and 800 ng/uL of salmon DNA in 6XSSC, 10% Dextran Sulfate. Suppression hybridization and washing were essentially as described in Klinger et al.
• coverslips containing extended DNA molecules were: 1X5m each in 2XSSC, 0.2XSSC, OJXSSC; 3X5m in 60% formamide in OJXSSC; 1X5 in 2XSSC; blocked with 3% BSA/4XSSC for 5m. All coverslips were washed at room temperature. Hybridization was detected with 0.5 ug/ml FITC-anti digoxigenin and 2.0 ug/ml Cy3- streptavidin.
• Photographs were taken directly from the microscope with Kodak Gold 400 film using FITC-Texas Reid (Omega Optical) or FITC-TRITC (Chroma Technology) dual band pass filters. Using this labelling and detection scheme, the expected pattern of fluorescence would be red-green-red or green-red-green.
• Hybridization was consistently detected on extended DNA molecules. In the more extended filaments, the signal appeared as a string of fluorescent spots, although a considerable variation in the degree of condensation among the hybridized molecules as well as within the molecule was noticed. In one trial, the entire length of the green-red-green pattern representing the 18 contig was about 40um; the expected length for 109kb of B-DNA is about 37um. The three cosmids could be uniquely identified in these preparations. Consistent patterns of fluorescence (representing the hybridized cosmids) were identified, especially where the cosmids met. Overlapping or abutting cosmids tended to run together, whereas non-fluoresence gaps were conspicuous.
• the three cosmids from the chromosome 21 contig were accurately resolved by hybridization to extended DNA molecules.
• the 2.3kb overlap and the 1.5kb gap could be identified in many of the filaments.

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• 1993
  • 1993-05-07 CA CA 2135072 patent/CA2135072A1/en not_active Abandoned
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