WO1989011545A1

WO1989011545A1 - Detection of the susceptibility to scrapie

Info

Publication number: WO1989011545A1
Application number: PCT/GB1989/000522
Authority: WO
Inventors: James Hope; Nora Hunter
Original assignee: Institute For Animal Health Limited
Priority date: 1988-05-17
Filing date: 1989-05-15
Publication date: 1989-11-30
Also published as: GB8811608D0; NZ229147A; AU3695789A; EP0418277A1; GB2235531B; AU619308B2; DK273990A; DK273990D0; GB2235531A; GB9023793D0

Abstract

It has been found that there are at least three polymorphic sites in the gene for PrP (the ''prion protein'') which are linked to the susceptibility of a bovine or ovine animal (especially a sheep) to scrapie (also known, in cattle, as bovine spongiform encephalopathy). In the non-coding 5' region, two such polymorphisms govern cleavage by EcoRI and HindIII respectively, and in the coding region, the third polymorphism changes codon 171 from CGG to CAG (arginine to glutamine). These polymorphisms can be detected by conventional assays using EcoRI, and HindIII, or equivalent enzymes, followed by a Southern blot; DNA sequence analysis, for example with PCR methods; or peptide sequence analysis.

Description

Detection of the susceptibility to scrapie.

The present invention relates to a method of detecting susceptibility of animals to a particular disease, namely scrapie.

Scrapie is an infectious disease of the central nervous system of sheep, goats and cattle. It is invariably fatal after very long incubation periods and is characterised by the presence of scrapie associated fibrils (SAF) in brain extracts of affected animals. Scrapie is transmissible to mice and hamsters and is usually studied in these animals. The disease known as bovine spongiform encephalσpathy (BSE) in cattle is almost certainly identical to scrapie and the term "scrapie" is used herein to cover BSE.

The glycoprotein PrP (molecular weight 33-35 000) is the major component of SAF which is expressed at the same level in normal and scrapie affected brain tissue. However, in the uninfected animal SAF do not form. Proteins of a similar molecular weight and highly conserved structure sequence are encoded by mouse and sheep genes although the ovine PrP mRNA (5 kb ) is about twice the length of the hamster and mouse PrP mRNA (2.5 kb).

Sinc with two alleles p7 and s7 is the major gene determining the incubation period of all strains of scrapie in mice. There is evidence that Sinc is linked to the PrP gene in inbred and congenic mouse strains from restriction fragment polymorphism (RFLP) analysis.

Development of scrapie in the natural host, sheep, also depends on genetic factors. The response of sheep to injection with SSBP/1 (a standard pool of scrapie strains) is mainly controlled by the gene Sip (analogous to Sinc). Sip has two alleles designated as Sip^SA and Sip^PA. When injected intra-cerebrally (i.e.) with SSBP/1, sheep whose genotype is sAsA or sApA have short incubation periods and pApA sheep have extremely long incubation periods (see Table 1). When SSBP/1 is given subcutaneously (s.c.) to young adult Cheviots, only those carrying the sA allele develop scrapie whereas pA homozygotes survive for a full lifespan.

An incubation test is time-consuming and may harm the animal in a commercially unacceptable way. There is therefore a need for a quicker and less harmful test, to be used to help plan breeding programmes.

It has been found that there is at least one restriction fragment length polymorphism (RFLP) associated with the sA and pA alleles.

One aspect of the invention provides a method of determining whether an ovine, caprine or bovine animal is susceptible to scrapie, the method comprising analysing the DNA of that animal for a polymorphism linked to scrapie susceptibility.

Preferably, the new method comprises:

(a) extracting DNA from the animal,

(b) digesting the DNA with EcoRI and/or HindIII or equivalent enzymes to obtain DNA fragments of a predetermined length, and

(c) analysing the resulting digest for DNA fragments of a predetermined length.

The DNA is usually, of course, extracted from material obtained from the animal, rather than being extracted directly from the animal.

The term "equivalent enzymes" covers enzymes which cleave at equivalent sites as EcoRI or HindIII. For example, given that HindIII recognises the sequence 5'-AAGCTT-3', it is clear that Alul and Oxal will have an equivalent action, as they recognise the sequence AGCT. Actual isoschizomers of HindIII include BbrI, Hin1731, Hinblll, HinJCII, HsuI and Mkil. The sequence recognised by EcoRI is 5'-GAATTC-3', and isoschizomers include RstI and SsoI. Candidate enzymes may be compared with EcoRI and Hindlll to ensure that they are sufficiently similar in their action not to generate spurious results. Preferably, the equivalent enzymes are actual isoschizomers, in other words they recognise exactly the same sequence and cut at exactly the same place. Clearly, the recognition of the same sequence is more important than cutting at exactly the same point, as a cut a few bases away from EcoRI or HindIII sites will have only an insignificant effect on the MW of the resulting fragments.

Conveniently, the DNA is extracted from blood, liver or brain tissue by a procedure which does not kill the animal or render it commercially useless.

The two enzymes of step (b) may be used simultaneously (or sequentially), but the resulting fragments are 3.4kb (Sip^{s As A} ) and 3.8kb (Sip^{P A P A} ) and are not so easily distinguished from one another.

Suitably, therefore, only one of the enzymes is used. The fragments which are generated may be detected by any suitable means. Suitable probes may be devised and constructed by known means, based on the sequence information relating to the PrP glycoprotein dislosed in Robakis et ai (1986) and Basler et al (1986). (See also Figure 4). The probe should hybridise in a useful way (i.e. with the stringency conditions being adapted to give a meaningful answer, as can be determined routinely and non-inventively) to the DNA encoding the PrP glycoprotein. This glycoprotein has been found to be highly conserved across the species and therefore DNA (preferably from the coding region) from, say, hamsters can be used to construct a probe for sheep DNA. A suitable probe is pEA974, available from Dr. N. Robakis, Institute for Basic Research in Development Disabilities, 1050, Forest Hill Road, Staten Island, N.Y. 10314, USA, although the performance of the invention does not rely upon the use of this particular probe. In general, the probe can represent any portion of the sequence lying between the two polymorphic sites shown in Figure 2. The probe hybridises to the region of DNA identified by the cross-hatched box in Figure 2, and it can be seen that 3.4 kb or 5.0 kb HindIII fragments or 4.4 kb or 6.8 kb EcoRI fragments will be detected, according to whether the allele is sA or pA, respectively. Clearly, if instead the probe bound to the region between the left-hand EcoRI site in Figure 2 and the polymorphic EcoRI site, the fragment which was characteristic of the polymorphism and which was detected would be a 2.4 kb fragment. Likewise a probe hybridising between the polymorphic HindIII site and the right-hand HindIII site would detect a 1.6kb fragment characteristic of the polymorphism. Thus, the analysis in step (c) may be for these fragments. Alternatively, the analysis may follow digestion with one or more further restriction enzymes, for example TagI following a HindIII digestion, yielding fragments which are indicative of the said EcoRI or HindIII fragments. The second enzyme site and the polymorphic first enzyme site should be on opposite sides of the DNA recognised by the labelled probe.

It should be noted that the RFLP is located in a non-coding portion associated with the gene for PrP.

It is to be understood that these fragment sizes are approximate and depend upon operational factors such as the (agarose) gel running conditions, source of standard DNA markers and so on. For example, under other conditions the 5.0 kb HindIII fragment appears to be only 4.8 kb long and the 6.8 kb EcoRI fragment appears to be 7.0 kb long. It is the relative mobility of the fragments which is important.

The term "hybridises" is used in the context of conditions which produce an informative signal within 70 days (5 × t½ of ³²P ). The probe is preferably at least 20 bases long. In a second embodiment of the invention, the polymorphisms giving rise to the presence or absence of the EcoRI and/or HindIII sites may be detected by differential hybridisation using allele specific oligonucleotides constructed according to the sequence information given herein and applied to DNA, RNA, chromosomes, cells or tissue sections, optionally following amplification, for example with the PCR method. For example, the oligonucleotide:ATC CTC TAT TAT GAA TTC TTC TGG AAA ACT (EcoRI site underlined) will bind more tightly to the DNA from the sA allele than to the DNA from the pA allele. Such a difference in hybridisation affinity can be detected by conventional techniques. Longer or shorter sequences of oligonucleotide may be devised non-inventively to suit the parameters of the particular hybridisation assay in order to maximise the sensitivity of the test; too short an oligonucleotide may be insufficiently specific for the polymorphic site, whereas too long an oligonucleotide may not show enough difference in binding affinity according to whether the EcoRI site is present or not. Differential hybridisation can be demonstrated by showing that an oligonucleotide imperfectly matched to the test nucleic acid forms a duplex which is denatured at a temperature lower than that required to denature a perfectly matched duplex. This temperature difference is commonly detected using radioactively labelled oligonucleotides with the test nucleic acid immobilised on membranes (ie. as in the Southern or Northern blot). Other methods available include the use of hydroxylapatite which binds double stranded nucleic acids, in situ hybridisation, and enzymes (eg. RNase A) which cut duplexes at mismatched regions, denaturing gradient gels, oligonucleotide ligation assays or indeed by direct sequencing. See also Landegren et al (1988) Science 242, 229-237). The polymerase chain reaction (PCR) may be used to amplify the DNA sequence which would include the polymorphic (eg EcoRI) site if present. (See Mullins et al, Cold Spring Harbor Symp. Quant.Biol. LI, 1986, pp263-273 and subsequent papers). The probe is made to the sheep PrP sequence and hybridises to the amplified DNA. Examples of PCR oligonucleotides (marked on Figure 5) for the EcoRI polymorphism are as follows :-

3' TAA ACA TTG AAA CGT ACA TGA ACA AAA CAC 5' 5' AAC CTT GAA AAC CAT TTG CAC CAT CTC AGT 3' Using these in a standard PCR reaction would generate an amplified fragment of 867 basepairs (bp). Cutting thus with EcoRI would give two fragments of about 567 and 300 bp if the site is present (pA linked) and would leave the fragment untouched when the site is absent ( sA linked). In the heterozygote, all three fragments would be seen. The fragments are best detected using the Southern procedure and a third oligonucleotide as a probe. This may be the same as the probe in the simple non-PCR method described above; it needs to lie between the primer oligonucleotides (for example spanning the EcoRI site) in order to work. The PCR system is rapidly becoming a standard technique and the enzyme required, Tag polymerase, is available commercially. Similarly, with respect to the HindIII polymorphism, genomic DNA can be cut with EcoRI and formed into a library in, for example, a lambda vector (e.g. lambda 1049). Clones are selected by positive hybridisation to a suitable probe (e.g. pEA974) and sequenced between the coding region and the 5' EcoRI site. This will then reveal the sequence of the polymorphism and thus enable oligonucleotides to be devised and used as above to detect the polymorphism. Alternatively, although more laborious, simple DNA or RNA sequencing can be used to Identify whether the polymorphism is present. Another polymorphism has been found by cloning and sequencing the two copies of the PrP gene from a Suffolk sheep heterozygous for the EcoRI polymorphism. In the protein coding regions of these gene copies there is a difference - a G to A change - which would result in codon 171 specifying arginine in the PrP protein molecule associated with the 4.4kb EcoRI fragment and a glutamine in that associated with the 6.8kb fragment. This polymorphism in the coding region may be detected by DNA sequence analysis or by differential hybridisation etc as detailed above or by sequence analysis of the polypeptide product. The PrP may be isolated by the methods given in Bendheim et al (1986), the EMBO Journal, 5(10), 2591-2597, and then sequenced by known peptide sequencing methods, for example using the ABI A470 protein sequenator with on-line h.p.l.c. analysis on a 120A pth-analyser (Applied Biosystems, Calif., USA).

The allele specific oligonucleotides needed to detect this new polymorphism in the sheep PrP gene are two strands of synthetic DNA each a perfect match for one of the polymorphic forms ie. differing only at one nucleotide (which shoould be internal to the sequence). Examples of these are:- (A) CAA GAC CAA TGA TAT GGC TAG GTG ACC AGA and (B) CAA GAC CAA TGA TAT GAC TAG GTG ACC AGA. Note:- These strands and their complementary strands will both work on DNA, whereas the complementary strands will work only on RNA . The length of the oligonucleotides could be different from A and B. They must however be long enough to hybridise specifically to the PrP sequence at the polymorphic region but short enough to be destabilised by the mismatched base.

A further aspect of the invention provides a kit for use in such test methods. The kit may comprise the required restriction enzyme and a (labelled) probe. Alternatively, the enzyme may be supplied separately (since many are widely available) so that the kit just comprises the probe. The kit is used with standard Southern biottins procedures. in the case of a kit for use in PCR procedures, the kit will comprise the PCR primer oligonucleotides and. optionally, a probe for use in Southern biottins procedures.

Preferred embodiments of the invention will now be described with reference to the accompanying drawings. fig 1: the result of a Southern analysis of Cheviot sheep DNA digested with HindIII (Figure 1A) or EcoRI (Figure 1B) and hybridised with ³²P-pEA974; Fig 2: a restriction map of the region of the sheep genome coding for the PrP glycoprotein; Fig 3: similar to Figure 1 but shows analysis of Blackface sheep DNA; Fig 4: (on 4 sheets): the partial nucleotide sequence of the hamster PrP gene; Fig 5 (on 15 sheets): the sequence of a large part of the sheep PrP gene, showing the polymorphic EcoRI site (at about base 4023) and candidate PCR oligonucleotides (at about 3720 to 3750 and about _4560 to 4590, underlined with wavy line).

Since 1961, our lines of Cheviot sheep have been selectively bred for increased (positive linei and decreased {negative line) incidence of scrapie in response to injection with SSBP/1. Negative line sheep are Sip^{P A P A} and positive line sheep are either Sip^{s As A} or Sin^{s A p}A. The Sip^{s A} allele is recessive in Cheviot sheep for the majority of known scrapie strains (the A-group strains), but dominant for the test inoculum, SSBP/1. There is also evidence that the Sip gene controls the incidence of natural scrapie in both Herdwick and Cheviot sheep, with the sA allele again acting with full dominance. in Swaleάale sheep lines the dominance of the "susceptibility" allele is only partial, heterozygotes having much longer scrapie incubation periods than homozygous susceptible sheet (Davieε and Kimbεrlin. 1 985) . In this specification, the phrase "susceptible to scrapie" is used to denote an animal which, following exposure to scrapie at an early state in its life, will develop clinical signs of the disease before reaching its normal age of death or the age at which it is normally slaughtered. Susceptibility is moreover used in relation to the "A" group of scrapie strains. With the "C" group, the relative resistance susceptibility rankings conferred by the sA or pA alleles may be reversed; the methods of the invention may nevertheless be useful in this context.

Example 1

High molecular weight DNA (in other words genomic DNA) was extracted from blood, liver or brain tissue of our Cheviot sheep using a modification of the method of Blin and Stafford (1976). Southern analysis (Maniatis et al., 1982) was carried out using as a probe pEA974, a cDNA clone made to hamster PrP mRNA (Robakis et al., 1982; Wu et, a., 1987). The sheep DNA was digested with restriction enzymes according to manufacturer's instructions, electrophoresed through a 1% agarose gel and blotted on to nitrocellulose membrane in 20 × SSPE (20 × SSPE is 3.6M NaCl, 200mM NaH₂ PO₄, 22mM EDTA pH 7.4). Membranes were prehybridised for at least 4 hours at 37° C in the following solution:- 5 × SSPE, 5 × Denhardts, 50% deionised formamide, 0.1% SDS and 100 ug ml^{- 1} sheared fish milt DNA. Hybridisation was in the same solution for 18h at 37° C with the addition of the insert fragment of pEA974 ( 8ng ml^-1) isolated from a low melting point agarose gel after digestion with EcoRI and HindIII . The probe was labelled with ³²P dCTP to 10⁹ c.p.s. per ug using the technique of Feinberg and Vogelstein (1984). Membranes were washed in 1 × SSPE, 0.1% SDS at 55° C and exposed to Kodak XAR-5 film at -70° C with a Lightning Plus intensifier screen for five days. Ten negative line and twenty one positive line sheep were tested. Two RFLP's were found with the enzymes EcoRI and HindIII and examples of these are shown in Figure 1. No such polymorphisms were found with BamHI, BstEII, EcoRV, KpnI, PstI, PvuII, SstI, TagI or Xbal.

In negative line sheep DNA a 5.0 kb HindIII fragment hybridised to pEA974 (Figure 1A tracks 1 and 2). Positive line sheep showed either a 3.4 kb HindIII fragment (tracks 6 and 7) or both the 5.0 and the 3.4 kb fragments (tracks 3, 4 and 5). With EcoRI, (Figure 1B) negative line sheep had a 4.4 kb fragment ( tracks 1 and 2 ) and positive line had either a 6.8 kb fragment (tracks 6 and 7 ) or had both the 4.4 and 6.8 kb fragments { tracks 3 , 4 and 5 ) . A preliminary restriction map constructed from our data is shown in Figure 2.

Evidence from the breeding records was examined in order to interpret these results. All ten animals from the negative line (all of which are Sip^PAPA) had only the 5.0 kb HindIII fragment. Of the positive line sheep, eleven were predicted to be Sip^{s A p A} heterozygotes on the basis of their pedigree and all of these showed both the 5.0 kb and the 3.4 kb HindIII fragments on Southerns. Of the ten animals predicted to be Sip^{s A} homozygotes six showed only the 3.4 kb HindIII fragment and four had both fragments (see Table 2). Given the difficulty of distinguishing Sip^{s A s A}, the data suggest that the 5.0 kb HindIII fragment can be regarded as a marker for pA and the 3.4 kb HindIII fragment as a marker for sA. As the EcoRI polymorphism of the PrP gene always corresponds to that seen with HindIII (Table 2), we also linked the 4.4 kb EcoRI fragment to the pA allele of Sip and the 6.8 kb EcoRI fragment to the sA allele. Example 2

Similar experiments were conducted with DNA from Blackface sheep. The results indicate that the same sized HindIII and EcoRI fragments hybridise to the hamster PrP clone (Figure 3) but that the pattern is not the same as in the Cheviots. Blackface 1 and 2 have the 5.0 kb HindIII and 4.4 kb EcoRI fragments which in Cheviots would indicate a Sip^{p A} homozygote. Blackface 3 has two HindIII fragments hybridising to the probe but only one (6.8 kb) EcoRI fragment. Blackface 3 (Sip^{s A p A} from the HindIII data or Sip^{s A s A} from the EcoRI data) would be designated 'positive line' if it was a Cheviot. In the light of this, it is of interest that animal 4 (which has the same pattern as Blackface 3) is a Blackface Cross which developed natural scrapie in the field.

Table 1. Incubation periods of SSBP/1 scrapie in Cheviot sheep.

Route of Incubation period (days ± SEM)* Negative line injection positive line (Sip^{p A p A})

(Sip^{s A s A} or Sip^{s A p A)}

i.c. 197 ± 7 917 ± 90 s .c. 313 ± 9 -

* Data adapted from Dickinson et al., 1968 Table 2. Linkage of PrP gene and Sip

Fragments Sip genotype predicted from breeding records hybridising to PrP cDNA pApA (10) ^a sApA (11) sAsA (10)

A. HindIII

5.0kb only 10^b 0

3.4kb only 0 0

Both 0 11

B. EcoRI

6.8kb only 0 0

4.4kb only 10 0

Both 0 11

^a Total numbers of sheep tested

^b Numbers of sheep FURTHER EXAMPLES OF SHEEP BREEDS

Additional developments are detailed below and summarised in Table 4. In order to facilitate understanding of the findings we have used a system similar to that used by Carlson et al to describe the mouse PrP RFLP forms. There are 4 forms of the sheep PrP gene described in Table 3.

Table 3 Forms of the sheep PrP gene defined by RFLP analysis

PrP Form Restriction fragment size (Kb)

Hindlll EcoRI A 5.0 4.4

B 5.0 6.8

C 3.4 6.8

D 3.4 4.4

Using this nomenclature, we have therefore established that our Cheviot sheep are : -

Negative line : Sip ^{P A P A} prp^{A / A}

Positive Line : Sip ^{s As A} prp^{c / c}

Sip ^{s A p A} PrP^{c / A}

1. NPU Cheviot sheep

a) Positive Line:- 42 animals tested: 14 are PrP^{c / c}

28 are PrP^{c / A} b) Negative Line:- 14 animals tested: 14 are prP^{A / A} 2. NPU Herdwick sheep

We have a small flock (about 30 animals) of Herdwick sheep selected in a similar manner to the Cheviot flock into a negative and positive line. We have tested 3 animals from each line.

a) Positive Line:- 2 are PrP^{c / A}

1 has both 4.4 + 6.8kb EcoRI fragments

b) Negative Line:- 1 is PrP^{A / A}

1 is PrP^{B / B}

1 has both 4.4 + 6.8kb EcoRI fragments

3. NPU Cheviot × Herdwick

A small number of Cheviot × Herdwick cross sheep were set up as part of an experiment formally to prove that the gene Sip acts in Herdwick sheep as it does in Cheviots. Two animals, when injected with SSBP/1, developed scrapie relatively quickly. Because of this and because of the way the cross was set up, these animals had to be Sip^{s A p A} heterozygotes. By RELP analysis, both these animals were PrP^{c / A} - exactly as Cheviot heterozygotes are.

4. Swaledale sheep

At Redesdale Experimental Husbandry Farm there is a flock of about 300 Swaledale sheep bred (from 1973) for reduced susceptibility to SW73 then SW75 scrapie sources (pooled homogenates of scrapie brains from different Swaledale flocks). This flock is therefore a negative line and is described in Davies and Kimberlin (1985).

We have collected blood samples from 29 animals (10% of the flock) and have been able to test all of these with one enzyme, EcoRI. Twenty one animals have one 4.4kb EcoRI fragment (in Cheviots this would identify Sip^{p A p A} animals); 8 animals have both 4.4kb and 6.8kb fragments. Hence 21 out of 29 negative line Swaledales have the same EcoRI fragment as the negative line Cheviots.

RELEVANCE TO NATURAL SCRAPIE

With natural scrapie, the sA allele of Sip is recessive, not dominant as with SSBP/1 experimental scrapie. Thus, animals contracting scrapie naturally should be Sip ^{s A s A} .

In our Cheviot positive line, 5 animals have developed natural scrapie. Four of these are PrP^{c / c} and one tested with only EcoRI had the 6.8kb fragment and must be PrP^B or PrP^c . In the Herdwick positive line one animal with natural scrapie had only the 6.8kb EcoRI fragment.

Six other natural scrapie cases from outside our flocks have also been tested (2 Cheviot, 2 Suffolk, 1 Charolais and 1 Blackface cross) and are detailed in Table 4. Of 12 cases examined so far, 10 have only the 6.8kb EcoRI fragment (the marker for Sip ^{s A s A} ), 1 has 2 EcoI fragments and 1 has the 4.4kb fragment only. This final animal was a Cheviot from the V.I. centre at St. Boswell and although clinically diagnosed as scrapie positive, was negative by pathology.

OTHER ANIMALS

Five out of six Anglo-Nubian goats were PrP^{B / c} , the remaining one being PrP^{c / c} . These goats are all susceptible to experimental scrapie. Table 4 RFLP Data

Hindlll EcoRI PrP

Animal Line Sip No 3.4kb 5.0kb 4.4kb 6.8kb group

Sheep

Cheviot³ + sAsA 14 + + C/C

+ sApA 28 + + + + C/A pApA 14 + + A/A

Herdwick³ + 2 + + + + C/A

+ 1 + +

- 1 + + A/A

- 1 + + B/B - 1 + +

Cheviot + sApA 2 + + + + C/A

Herdwick

Cross^a

Swaledale^b - 21 + 8 + +

Blackface^c 2 + + A/A

1 + + + B/C

Suffolk^d 3 + +

Suffolk^a 3 +

Suffolk^a 1 + + HindIII EcoRI PrP

Animal Line Sip No 3.4kb 5.0kb 4.4kb 6.8kb Group

Natural

Scrapie

Cheviot a + sAsA 4 + + C/C

³ + 1 + e 1 + f 1

Herdwick^a + 1 + Suffolkf 1 + Suffolk ^f 1 + Charolais^f 1 +

Blackface Cross^f 1 + + + B/C

Goats^a 5 + + B/C

1 + + C/C

Cow^a B/B

Sources of animals : ^a - AFRC/MRC Neuropathogenesis Unit, Edinburgh; ^b - Redesdale; ^c - Moredun; d - Dingwall; e - Thornhill; ^f - St Boswell.

References

Basler et al, Cell (1986), 46, 417-428.

Blin, N. and Stafford, D.W. (1976) Nucleic Acids Research

9., 2303.

Carlson, G.A. et aJ (1988) Mol.Cell.Biol. 8(12), 5528- 5540. Dickinson, A.G., Meikle, V.M.H. and Fraser, H. (1986)

Journal of Comparative Pathology 78, 293.

Feinberg, A.P. and Vogelstein, B. (1984) Analytical

Biochemistry 137, 266.

Maniatis, T., Fritsσh, E.F. and Sambrook, J. (1982)

Monecular Cloning: A Laboratory Manual. New York: Cold

Spring Harbor Laboratory.

Robakis, N.K., SAWH, P.R., Wolfe, G.C., Rubenstein, R.,

Carp, R.I. and Innis, M.A. (1986) Proceedings of the

National Academy of Sciences, U.S.A. 83, 6377.

WU, Y., Brown, W.T., Dobkin, C, Devine-Gage, E., Merz,

P. and Wisniewski, H.M. (1987) Nucleic Acids Research 15,

3191.

Claims

1. A method of determining whether an ovine, caprine or bovine animal is susceptible to scrapie, the method comprising analysing material from that animal for a polymorphism linked to scrapie susceptibility.

2 A method according to Claim 1, the method comprising

(a) extracting DNA from the animal,

(b) digesting the DNA with an enzyme to obtain fragments of a predetermined length, and

(c) analysing the resulting digest for DNA fragments of a predetermined length.

3. A method according to Claim 2 wherein the enzyme is EcoRI, HindIII or an equivalent of either.

4. A method according to Claim 3 wherein step (b) comprises a single digestion with only one of the two enzymes and, in step (c), the analysis comprises looking for approximately 3.4 kb or 5.0 kb HindIII fragments or for approximately 4.4 kb or 6.8 kb EcoRI fragments, or for fragments indicative of any of the said fragments.

5. A method according to Claim 3 or 4 wherein, in step (c), the analysis comprises hybridising the digest with a labelled probe known to hybridise to DNA associated with the PrP gene.

6. A method according to Claim 5 wherein the probe hybridises to part of the PrP coding region.

7. A method according to Claim 5 wherein the probe hybridises to a region of the PrP glycoprotein coding region between the left-hand EcoRI site and the right-hand HindIII site shown in Figure 2.

8. A method according to any one of Claims 2 to 7 wherein the DNA is first amplified by means of a polymerase chain reaction.

9. A method according to Claim 1 wherein the said material is DNA, RNA or polypeptide and the method comprises identifying the sequence change associated with the polymorphism.

10. A method according to Claim 9 wherein the method comprises using differential hybridisation to DNA or RNA, or sequencing DNA, RNA or polypeptide.

11. A kit for determining whether an ovine, bovine or caprine animal is susceptible to scrapie, the kit comprising an enzyme which will cleave DNA at a polymorphic site associated with the sA allele of the Sip gene and/or means to detect the presence or absence of the polymorphism.

12. A kit according to Claim 11 wherein the detection means Is a probe which hybridises to DNA encoding PrP glycoprotein .

13. A kit according to Claim 12 wherein the probe will hybridise to a region of the DNA encoding PrP glycoprotein between the left-hand EcoRI site and the right-hand HindIII site of Figure 2.

14. A kit according to Claim 11, 12 or 13 wherein the probe is pEA974.

15.. A kit according to any one of Claims 10 to 13 wherein the probe is chemically or physically labelled for recognition.

16. A kit according to any one of Claims 11 to 15 comprising^' the restriction enzyme EcoRI and/or HindIII.