DIAGNOSTIC TECHNIQUE

Abstract: A method of diagnosis suitable e.g. for differentiating between hairs of different animals, is adapted to determine the source of a biological structure (e.g. the hair) by hybridising a species-specific probe to single stranded genetic material in situ within said structure whilst maintaining, or substantially maintaining, the integrity thereof. Double stranded genetic material within the structure may be accessed and rendered single stranded, groups are rendered unreactive that react with a polynucleotide probe other than likewise reactive groups comprised by the genetic material, the probe is hybridised with the single stranded genetic material and the structure screened for hybridisation.

Description: DIAGNOSTIC TECHNIQUE This invention relates to methods of diagnosis which use molecular biology techniques. Poly - or oligonucleotide probes (hereinafter "probes") comprising a nucleotide sequence which is hybridisable with RNA or single stranded DNA (hereinafter "genetic material") within a cell or specimen are routinely used in diagnosis. The degree of preparation of the genetic material varies, but typically involves its isolation from the cell containing it, and frequently, a variable amount of cleavage and electrophoretic separation. It is not absolutely necessary to isolate genetic material from cells prior to screening with probes. Indeed, prokaryotes may simply be washed, or otherwise placed, onto a support and then subjected to treatments which lyse them jj situ to expose genetic material. These cells have a relatively high genetic material content, and notwithstanding the relative simplicity of technique accruing from dispensing with isolation of the material proper, it is preferred that at least the protein component of the cells is removed before application of the probe. Substantial and/or variable losses in genetic material occur in textile processing and during its isolation, and significant such losses in material occur even when just the protein component is removed from cells. The problem of substantial and/or variable loss of genetic material from a textile, for example, which comprises more than a single type of fibre means that specific ratios of the fibres in blends thereof cannot be determined by hitherto used molecular-biology techniques. The present invention provides a method of diagnosis using molecular biology techniques which, at least to some extent, overcomes the disadvantages characterised by prior such methods. According to the present invention there is provided a method of diagnosis adapted to determine the source of a biological structure, comprising hybridising a species-specific probe to single stranded genetic material in. situ within said structure whilst maintaining, or substantially maintaining, the integrity thereof. The genetic material may be DNA. The method may comprise the steps of: accessing double stranded genetic material within the structure, rendering said genetic material single stranded, rendering unreactive groups within the structure that react with a polynucleotide probe other than likewise reactive groups comprised by said genetic material, hybridising said probe with the single stranded genetic material and screening the biological structure for hybridisation. The structure may be multi-cellular, and may, for example, be hair or fur. Such hair or fur may be from quadrupeds such as the cashmere goat, yak, and llama, for example. The genetic material in the structure may be DNA which may be accessed by a limited digestion of the structure with proteinases. The genetic material may be accessed by immersing the structure in an aqueous solution. The solution may comprise a reducing agent which may be 2-mercaptoethanol or dithiothreitol. The solution may comprise ethylenediaminetetra- acetic acid (EDTA). The solution may comprise a detergent which may be sodium or lithium dodecyl sulphate. Alternatively, or additionally, the genetic material may be accessed by sonicating or otherwise mechanically agitating the structure. The genetic material may be rendered single stranded by known means including denaturing same by incubating the structure in alkali or by subjecting it to molecular excitation, as for example, by microwave irradiation. Groups (hereinafer "said groups") within the structure which react with the polynucleotide probe, other than likewise reactive groups comprised by said genetic material, may be rendered unreactive by conventional means including incubating the structure with sonicated salmon testes DNA and/or urea. The positive charge density on said groups may be reduced by increasing the pH within the structure. The polynucleotide probe may be attached to an enzyme which is chosen having regard to the use of known enzyme-linked reactions whereby ultimately a substrate may be converted into a quantifiable and/or quantitatabl product. The probe may be conjugated to a protein. Following hybridisation, the structure may be incubated with antibodies which are directed against the protein, and which are conjugated to an enzyme which converts a substrate into a quantifiable and/or quantitatable product. The probe may be conjugated to a fluorescent dye Specific probes may be conjugated with specific fluorescent dyes and thus a plurality of probes conjugated to dyes having a plurality of fluorescence maxima may be provided, whereby the fluorescence maxima are used to identify the probes when they are located within structures. The fluorescent dyes may, for example, be rhodamine, fluorescein, dansyl chloride, fluorescamine, fluoreceinamine, sulphurhodamine, tetramethylrhodamine- isothiocyanate, and fluorescent derivatives thereof. Hybridisation of a radioactive probe to the single stranded genetic material may be determined autoradiographically. Hybridisation may be determined by an enhanced chemiluminescent technique, in which the probe is cross linked to horseradish peroxidase which in the presence of hydrogen peroxide converts luminol into 3-aminophthal- ate. In the presence of enhancers the aminophthalate exhibits an emission maximum at 428 n which can be detected by film sensitive to blue light. The probe may be RNA or single stranded DNA obtained directly, or otherwise derived, from said quadrupeds. The probe may be synthetic. The synthesis may recognise the expression of a particular gene or genes in the cells comprised by the structure. The particular gene or genes may encode keratins. Gene polymorphism may be considered in the preparation of the probe, so that a multiplicity of different probes may be generated so as to be capable of hybridising with genetic material encoding ostensibly a single family of proteins. The probe may likewise be synthesised having regard to the genome of the animal from which the structure is derived. The invention will be further apparent from the following description, which illustrates the technique according to the invention, by way of example only, and with reference to the detection of goat-derived textile fibres in a fabric comprising a blend of fibres, some derived from goat and others from yak. The technique for detecting species specific genetic material in, situ within a multi-cellular structure, comprises a plurality of steps. The DNA within the putative goat fibre is accessed by a limited digestion thereof with proteinases such as the fungal type XI proteinase from Tritirachium album, and the fibre is typically incubated in a solution comprising between 0.1 and 0.5% (w/v) thereof depending on the desired result. It will be appreciated that other proteinases can be used, depending on the identity of the biological structure and degree of access required. Under appropriate conditions the solution contains a reducing agent such as 2-mercaptoethanol or dithiothreitol. The solution can also contain EDTA and/or a detergent such as the sodium or lithium salt of dodecyl sulphate. The detergent is present at 0.1 to 1% (w/v) and the fibre is incubated therein typically overnight. Alternatively, or additionally, the DNA might be accessed by sonicating or otherwise mechanically agitating the fibre. The DNA within the fibre is rendered single stranded by denaturation in alkali, although other techniques including subjecting it to molecular excitation, as for example, by microwave irradiation may, under certain circumstances, be more appropriate. Positively charged groups on, for example, amino acids, such as the guanidino group on arginine or epsilon amino group on lysine, may react with the negatively charged poly or oligo nucleotide probe, resulting in high backgrounds. These groups are relatively deactivated by incubating the fibre with sonicated salmon testes DNA and/or urea. Alternatively and/or additionally the positive charge density on the groups is reduced by increasing the pH within the fibre. A probe is prepared comprising a polynucleotide sequence which is complementary to a sequence of single stranded DNA within the fibre. The probe comprises single stranded DNA obtained directly, or otherwise derived, from the species of goat from which the fibre is derived. It will be appreciated, however, that a synthetic probe can equally well be used and may have the advantage that its synthesis may take cognizance of the expression of a particular gene or genes in the cells comprised by the fibre. These genes may for example encode keratins, and the probe may likewise be synthesised having regard to the genome of the animal from which the fibre is derived. However the probe is obtained it is rendered radioactive (for example in a nick translation system) and hybridised with the DNA within the fibre. Suitable washing regimes are performed and the hybridisation detected autoradiographically. Where the fibre is derived from other than the species of goat aforementioned, the DNA therein will not hybridise with the probe specific for the goat species and no, or little, radiation will be detected by the X-ray film used in the autoradiographic step. In view of the foregoing, however, it will be now appreciated that the probe need not neccessarily be radioactive, but might be attached to an enzyme which is chosen having regard to the use of known enzyme linked reactions whereby ultimately a substrate is converted into a quantifiable and/or quantitatable product. The probe could be conjugated to a protein. Following hybridisation, the fibre may be incubated with antibodies which are directed against the protein, and which are conjugated to an enzyme which converts a substrate into a quantifiable and/or quantitatable product. The probe may be conjugated to a fluorescent dye, Specific probes may be conjugated with specific fluorescent dyes and thus a plurality of probes conjugated to dyes having a plurality of fluorescence maxima may be provided, whereby the fluorescence maxima are used to identify the probes when they are located within fibres. The fluorescent dyes may, for example, be rhodamine, fluorescein, dansyl chloride, fluorescamine, fluoreceinamine, sulphurhodamine, tetramethylrhoda ine isothiocyanate, and fluorescent derivatives thereof. Thus a probe specific for yak DNA may be labelled with rhodamine which fluoresces red under UV light, and a second probe specific for goat DNA may be labelled with fluorescein which fluoresces green when subjected to UV light so that yak and goat derived fibres may, after the hybridisation step, be recognised simply from the colour of fluorescence as visualised under a UV microscope. Alternatively the hybridisation may be determined by an enhanced chemiluminescent technique, in which the probe is cross linked to horseradish peroxidase which in the presence of hydrogen peroxide converts luminol into 3-aminophthalate. In the presence of enhancers the aminophthalate exhibits an emission maximum at 428 nm which can be detected by film sensitive to blue light. It will be appreciated that it is not intended to limit the invention to the above example only, many variations, such as might readily occur to one skilled in the art, being possible, without departing from the scope thereof as described herein. For example, the genetic material within the structure under test may be amplified by any known means including the polymerase chain reaction method.

Claims: CLAIMS 1. A method of diagnosis adapted to determine the source of a biological structure, comprising hybridising a species-specific probe to single stranded genetic material Lτ__ situ within said structure whilst maintaining, or substantially maintaining, the integrity thereof. 2. A method according to claim 1, in which the genetic material comprises DNA. 3. A method according to claim 1 or claim 2, comprising the steps of: accessing double stranded genetic material within the structure, rendering said genetic material single stranded, rendering unreactive groups within the structure that react with a polynucleo¬ tide probe other than likewise reactive groups comprised by said genetic material, hybridising said probe within the single stranded genetic material and screening the biological structure for hybridisation. 4. A method according to any one of claims 1 to 3, in which the structure is multicellular. 5. A method according to claim 4, in which the structure is hair or fur. 6. A method according to claim 5, in which the hair or fur is from a quadruped. 7. A method according to claim 6, used to distinguish between hair of different quadrupeds such as cashmere goat, yak and llama. 8. A method according to claim 2, in which the DNA is accessed by a limited digestion with proteinases. 9. A method according to any one of claims 1 to 8, in which the genetic material is accessed by immersing the structure in an aqueous solution. 10. A method according to claim 9, in which the said solution comprises a reducing agent. 11. A method according to claim 10, in which the reducing agent comprises 2-mercaptoethanol. 12. A method according to claim 10 or claim 11, in which the reducing agent comprises dithiothreitol. 13. A method according to any one of claims 9 to 12, in which the solution comprises ethyldiaminetetra-acetic acid (EDTA). 14. A method according to any one of claims 9 to 13, in which the solution comprises a detergent. 15. A method according to claim 14, in which the detergent comprises sodium or lithium dodecyl sulphate. 16. A method according to any one of claims 1 to 15, in which the genetic material is accessed by sonicating or otherwise mechanically agitating the structure. 17. A method according to any one of claims 1 to 16, in which the genetic material is rendered single-strande by denaturising the same by incubating the structure in alkali. 18. A method according to any one of claims 1 to 17, in which the genetic material is rendered single-strande by molecular excitation as by microwave radiation. 19. A method according to any one of claims 3 to 7, in which said groups within the structure that react with the polynucleotide probe are rendered unreactive by incubating the structure with sonicated salmon testes DNA and/or urea. 20. A method according to claim 19, in which the positive charge density on said groups is reduced by increasing the pH within the structure. 21. A method according to claim 3, in which the polynucleotide probe is attached to an enzyme which is chosen having regard to the use of known enzyme-linked reactions whereby ultimately a substrate is converted into a quantifiable and/or quantitatable product. 22. A method according to claim 3, in which said probe is conjugated to a protein. 23. A method according to claim 22, in which, following hybridisation, the structure is incubated with antibodies which are directed against the protein and which are conjugated to an enzyme which converts a substrate into a quantifiable and/or quantitatable product. 24. A method according to claim 3, in which the probe is conjugated to a fluorescent dye. 25. A method according to claim 24, in which a plurality of probes conjugated to specific fluorescent dyes are provided and the fluorescence maxima are used to identify the probes when they are located within the structure. 26. A method according to claim 24 or claim 25, in which the fluorescent dye or dyes is or are selected from rhodamine, fluorescein, dansyl chloride, fluorescamine, fluoreceinamine, sulphurhodamine, tetramethylrhodamine-isothiocyanate, and fluorescent derivatives thereof. 27. A method according to any one of claims 1 to 28, in which hybridisation of a radioactive probe to the single stranded genetic material is determined autoradio graphically. 28. A method according to any one of claims 1 to 27, in which hybridisation is determined by an enhanced chemiluminescent technique in which the probe is cross linked to horseradish peroxidase which in the presence of hydrogen peroxide converts luminol into 3-aminophthalate. 29. A method according to any one of claims 1 to 28, in which the probe is RNA or single stranded DNA. 30. A method according to any one of claims 1 to 29, in which the probe is synthetic. 31. A method according to claim 30, in which the synthesis recognises the expression of a particular gen or genes in the cells comprised by the stucture. 32. A method according to claim 31, in which the said particular gene or genes encodes keratins. 33. A method according to any one of claims 30 to 32, in which a multiplicity of different probes is generated so as to be capable of hybridising with genetic material encoding ostensibly a single family of proteins, taking into account gene polymorphism. 34. A method according to any one of claims 30 to 33, in which the probe is synthesised having regard to the genome of an animal from which the structure is derived.

Inventors: HAMLYN PAUL, MCCARTHY BRIAN JOSEPH, NELSON GORDON, HAMLYN, PAUL, MCCARTHY, BRIAN, JOSEPH, NELSON, GORDON
Applicants: BRITISH TEXTILE TECH, BRITISH TEXTILE TECHNOLOGY GROUP
Application year: 1991
Main classification: G01N33
IPC codes: C12Q1, G01N33