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• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

• the present invention relates to a method for digesting cells or tissues in order to provide a solution comprising DNA in a form suitable for hybridisation.
• Prior art methods for preparing cellular DNA in a form suitable for hybridisation involve a complex series of steps including disruption of the cells, enzymatic digestion of various macromolecules, precipitation, separation and dissolution steps in order to remove unwanted cellular components and leave a more or less pure solution of DNA. Reagents used in this procedure may interfere with hybridisation and must also be removed. A typical example of such a process can be found in Blin, N. and Stafford, D. . 1976, Nucleic Acids Research, 3_, 2303.
• Brandsma and Miller Proc. Natl. Acad. Sci. USA; 77 (11), 6851-6855, (1980) and Wagner e_t al. , Identification of Human Papillomavirus in Cervical Swabs by Deoxoyribonucleic Acid j Situ Hybridisation, Obstetrics and Gynaecology 6 ⁇ , 767-772 (1984) describe method for detecting DNA in un-fractionated cells but these methods are probably not sensitive enough for the detection of single copy genesin less than 10 5 cells.
• the method of Brandsma and Miller is carried out on a nitrocellulose filter and comprises treating the cells with 0.5 M sodium hydroxide for 7 minutes at ambient temperature and washing with neutralising buffers and solvents. Such a process partially digests the cell contents, releasing DNA, but will not destroy cellular macromolecules sufficiently to prevent interference with a hybridisation test.
• the present invention provides a process for producing a solution of cellular DNA from cells or tissue which process consists of treating the cells or tissue with a strongly alkaline aqueous medium for a time and at a temperature sufficient to form a solution suitable for hybridisation for instance a clear solution.
• Solutions ⁇ f DNA prepared according to the present invention may be used for hybridisation by application directly to a suitable filter support. Alternatively the solution may be used, after neutralisation of the excess hydroxide ions for analysis by hybridisation directly in solution, by enzymatic amplification of target sequences by the DNA polymerase chain reaction (R.K. Saiki et al, Nature, 324, , 163 (1986)) or by similiar methods.
• DNA is more resistant to degradation by strongly alkaline media than any other cellular acromolecule and the present invention exploits the differential rates of such degradation.
• the process of the invention will normally be applied to whole (live or fixed) cells or tissues but is equally applicable to disrupted cells.
• the benefits of simplicity of procedure and of sensitivity are lost if separation steps precede the digestion in hydroxide medium.
• concentration of hydroxide ions in the aqueous medium which is significant in achieving the required degradation and the nature of the cation is less critical.
• concentration of hydroxide ions, the duration of the reaction and the temperature at which the reaction is conducted may be varied as convenient though it will be appreciated that less concentrated solutions of hydroxide ions will require longer time and/or higher temperatures; shorter times can be achieved using more concentrated hydroxide ions and/or higher temperatures and lower temperatures are possible when using more concentrated hydroxide ions and/or a longer duration of reaction.
• the temperature of the reaction should be above the freezing point and at, or preferably below, the boiling point of the reaction mixture.
• the reaction is performed at from about 20°C to about 95°C, more preferably at about 80°C.
• the concentration of hydroxide ions is conveniently from about 0.1M to about IM, more preferably from about 0.25M to about 0.5M and most preferably about 0.4M. Such concentrations may be achieved using a variety of alkaline or basic materials, preferably using an alkali metal or alkaline earth metal hydroxide, more preferably sodium or potassium and most preferably sodium hydroxide.
• the duration of the reaction is selected having regard to any constraints on the temperature and/or concentration of hydroxide ions to be used.
• the duration will be in the range of from a few minutes to a few hours preferably from 5 minutes to 1 hour.
• a duration of, for instance, the order of half an hour, particularly 30 minutes is convenient but a reaction time of about 10 minutes is considered optimal.
• the cells or tissue are digested using 0.4M sodium hydroxide at a temperature of about 80°C for about half an hour. This affords a solution containing DNA suitable for hybridisation in denatured form and which will generally have been degraded only to limited extent so that it is still suitable for analysis by hybridisation.
• the paraffin wax may be dissolved using a solvent such as xylene and then ethanol.
• Solutions of DNA produced according to the present invention may be used for hybridisation according to a variety of techniques.
• the DNA is bonded to a substrate such as a nitrocellulose or, preferably, nylon membrane to which the DNA becomes convalently bound (Reed, K.C. and Mann, D.A, (1986) Nucl. Acid, Res 13, 7207-7221).
• a substrate such as a nitrocellulose or, preferably, nylon membrane to which the DNA becomes convalently bound
• conventional hybridisation methods may be applied. Because the DNA preparation is a simple one-step process the losses of material are minimised, it is possible to work with very small samples and the absence of interference from extraneous cell debris permits detection of a single copy of a DNA sequence per cell in a sample of only 10 4 cells. This in turn allows the detection of, for instance, viral DNA from human immunodeficiency virus (HIV), hepatitis virus etc. in blood samples and mycoplasma contamination of cell cultures with greater sensitivity than previously possible.
• HIV human immunodeficiency virus
• the invention also provides a process for producing a substrate having cellular DNA bound thereto which process comprises treating cells or tissue with a strongly alkaline aqueous medium for a time and at a temperature sufficient to produce a solution of DNA suitable for hybridisation and contacting a hybridisation substrate with the solution under conditions permitting DNA to bind to the substrate.
• the substrate is a nylon membrane in which case the binding is suitably conducted in the presence of about 0.4M sodium hydroxide and at about 20°C.
• the DNA solution is produced by treatment of cells with 0.4M sodium hydroxide at 80°C for about 10 minutes and the solution, after cooling to about 20°C, is applied directly to the nylon hybridisation membrane which, after binding the DNA, is washed with neutralising buffer. Hybridisation may then be conducted according to standard techniques.
• the present invention further provides a process for detecting a DNA sequence within cells suspected to contain such DNA which comprises producing an aqueous solution of DNA by treating the cells with a strongly alkaline aqueous medium for a time and at a temperature sufficient to produce a solution of DNA suitable for hybridisation and contacting the DNA thus produced with a probe, comprising nucleic acid hybridisable to the DNA sequence to be detected and a detectable label, under conditions permitting hybridisation between the DNA sequence to be detected and the probe.
• the DNA solution produced in the first step is neutralised with suitable acid or buffer solution and hybridisation is conducted in the solution thus obtained.
• a suitable acid or buffer solution in which the DNA solution produced in the first step is neutralised with suitable acid or buffer solution and hybridisation is conducted in the solution thus obtained.
• hybridisation substrate is contacted with the solution under conditions permitting DNA to bind to the substrate, the substrate is washed and hybridisation is conducted by contacting the substrate with a solution of the probe.
• the probe and label may be any probe/label combination, of which a large number are readily available.
• One advantage of the process of the present invention is that interference in the hybridisation by extraneous cell debris is minimised or eliminated.
• the hybridisation substrate can be washed to remove residues from the cells other than the DNA before hybridisation and the substrate with bound DNA is therefore suitable for re-use in further hybridisation experiments optionally after removal of the first probe.
• BHK cells suspended in 50ul of PBS were made up to 250ul with 0.5M NaOH resulting in a final NaOH concentration of 0.4M. After heating to 80°C for 30 minutes, duplicate samples were aliquoted onto Genescreen plus as above and the filter was probed with pCADl42 cDNA (6.5 kb) (Shigesada et ⁇ al. , Mo . Cell. Biol., 5_, 1735-1742 (1985)). labelled by random priming as described below.
• Figure At Detection of pUC13 DNA in the presence of normal BHK cells 2.5 x 10 4 BHK cells suspended in 50ul of PBS were made up to 250ul with 0.5M NaOH resulting in a final NaOH concentration of 0.4M.
• SiHa and C331 cells were suspended at 2.5 x 10 4 cells in 50ul of PBS and were hydrolysed in 250ul 0.4M NaOH at 80°C for 30 minutes then duplicate samples were aliquoted onto Genescreen plus as above and the filter was probed with HPV16 DNA labelled by random priming as described below.
• 1 x SSPE 150mM sodium chloride, 15mM sodium phosphate, pH 8.
• Genescreen plus membrane was from NEN. All cells were grown in Dulbecco's modified Eagles medium (E4). C331 (Auersperg N, J. Nat. Cancer Inst. 3_2, 135-164, 1964) and SiHa (Friedl F e_t al ⁇ Proc. Soc. Exp. Biol. Med. 135, 543-545, 1970) are human cervical carcinoma cells kindly supplied by Dr Lionel Crawford. HeLa DNA was prepared by the Proteinase K method (Maniatis e_t al. , "Molecular Cloning, a Laboratory Manual” ColdSpring Harbour Laboratory (1982)). BHK cells are baby Syrian hamster kidney cells. DNA probes were prepared by random priming (Feinberg and Vogelstein, Anal. Bioche .
• RNA probes were prepared using the SP6 promoter/polymerase system (SP6 RNA polymerase was from Promega Biotech) and labelled UTP (400 Ci/mmol, Amersham) according to the methods of Melton et al., Nucl. Acid. Res. 12, 7035-7056 (1984)) after subcloning DNA fragments into plasmids pSP64 or pSP65.
• SP6 RNA polymerase was from Promega Biotech
• UTP 400 Ci/mmol, Amersham
• the solution was drawn through by capillary action when a capillary type slot blotter was used (relying on capillary action or absorbent towels).
• Wells were rinsed with lOOul of the 0.4M NaOH solution, the apparatus dismantled and the filter placed in 250ml of 2x SSC for 5 minutes to neutralise it. The filter was blotted and allowed to dry at room temperature for at least one hour before use.
• the filter was evenly wetted with 2 x SSC and placed in a suitable container or thick plastic bag.
• Prehybridisation was for at least 15 minutes at 42°C in 50% deionised formamide, 5 x SSPE, 5 x Denhardt's 1% SDS, lOOug/ml sheared and denatured herring or salmon sperm DNA after removal of air bubbles.
• the probe was heated to 65°C for 10 minutes and cooled on ice before adding it to the filter.
• Hybridisation was in the same solution containing up to 10 7 dpm/ml of labelled denatured probe at 42°C (on a rocker if available) for 2-16 hours after removal of air bubbles. Probe was removed and the filter washed as follows: 5—15 minutes in 250ml of 2 x SSC at room temperature, 30 minutes in 250ml of 2 x SSC, 1% SDS at 65°C and finally, if high stringency was required, 30 minutes in 250ml of 0.2 x SSC, 1% SDS at 65°C. Excess buffer was blotted off on 3MM paper but the filter was not dried if it was intended to strip off the probe and reuse the blot. For autoradiography, the filter was wrapped in plastic.
• probe was removed by boiling in ImM EDTA pH 8.0, 1% SDS for 30 minutes. Autoradiography was carried out at -70°C with intensifying screens using Kodak XAR5 film, preflashed to give a linear response.
• Figure 2 shows that using Yeast whole RNA, essentially no RNA - dependant signal remains after hydrolysis for 5 minutes in 0.4M NaOH at 80°C.
• the standard conditions initially adopted for quantitating gene copy number were to hydrolyse cells in 0.4M NaOH at 80°C for 30 minutes. After further investigation of the reaction time an optimal duration of 10 minutes was adopted.
• CAD CAD
• 6.5Kb of sequences corresponding to the amount of CAD cDNA in the probe could be detected in as few as 5 x 10 3 cells and corresponding to 30fg of target DNA.
• Amplified genes of interest in cells can certainly be detected in fewer than 10 4 cells although the practical limit has not yet been determined.
• the limits of the sensitivity for detection of a plasmid sequence diluted in mammalian cells was determined using pUCl3 DNA diluted in BHK clone 9.4 cells (10 4 cells per slot). 25fg of the plasmid DNA could be detected readily using a DNA probe, approaching the lower limit of 3 fg claimed by Church and Gilbert Proc. Natl. Acad. Sci 81, 1991-1995 (1983)) for RNA/DNA hybridisation. It is possible that by changing the specific activity of the probe or the hybridisation and washing conditions that this could be improved. About 5 x 10 5 cells can be loaded per slot before saturating the support.
• the main advantage of this technique is that is enables the quantification of DNA sequences in cells by nucleic acid hybridisation without the need to first isolate pure DNA. Taken together with the high sensitivity of the hybridisation to nylon membrane, this affords a considerable improvement in the ability to detect sequences in very small numbers of cells as well as an improved ability to handle large numbers of samples.
• a short list of other uses would include the analysis of gene copy number in gene amplification, detection of DNA viruses and pro-retroviruses in tissues and cells lines, detection of transfected DNA sequences in cell lines and transgenic animals and DNA-typing blood parasites such as Plasmodium falciparum in humans and Babesia bovis in cattle.
• a single 5 to 10 micron section of approximately lcm 2 area (or equivalent number of sections to approximate to this tissue volume) of formalin fixed paraffin embedded tissue is sectioned and placed in an Eppendorf tube.
• the paraffin wax is removed from the tissue by washing in xylene and then in absolute ethanol.
• the tissue is then hydrolysed in 0.4M sodium hydroxide for one hour. This time is optimal for this type of sample.
• the supernatent is then pipetted on to a nylon membrane [Gene Screen Plus] though a slot or dot blot manifold, washed with 0.4M NaOH and rinsed in 2 x SSC before being probed with radiolabelled DNA.
• Hybridisation, washing and autoradiography are carried out according to Example 1.

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• 1988
  • 1988-07-21 EP EP88906000A patent/EP0377573A1/de not_active Withdrawn
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