WO1989011545A1 - Detection de sensibilite a l'encephalopathie spongiforme bovine - Google Patents
Detection de sensibilite a l'encephalopathie spongiforme bovine Download PDFInfo
- Publication number
- WO1989011545A1 WO1989011545A1 PCT/GB1989/000522 GB8900522W WO8911545A1 WO 1989011545 A1 WO1989011545 A1 WO 1989011545A1 GB 8900522 W GB8900522 W GB 8900522W WO 8911545 A1 WO8911545 A1 WO 8911545A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- dna
- ecori
- prp
- hindiii
- probe
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Classifications
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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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- 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.
- SAF scrapie associated fibrils
- BSE bovine spongiform encephal ⁇ pathy
- 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.
- RFLP restriction fragment polymorphism
- RFLP restriction fragment length polymorphism
- 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.
- the new method comprises:
- the DNA is usually, of course, extracted from material obtained from the animal, rather than being extracted directly from the animal.
- equivalent enzymes covers enzymes which cleave at equivalent sites as EcoRI or HindIII.
- HindIII recognises the sequence 5'-AAGCTT-3'
- 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.
- the equivalent enzymes are actual isoschizomers, in other words they recognise exactly the same sequence and cut at exactly the same place.
- 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.
- the DNA is extracted from blood, liver or brain tissue by a procedure which does not kill the animal or render it commercially useless.
- 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.
- 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.
- 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.
- the fragment which was characteristic of the polymorphism and which was detected would be a 2.4 kb fragment.
- a probe hybridising between the polymorphic HindIII site and the right-hand HindIII site would detect a 1.6kb fragment characteristic of the polymorphism.
- the analysis in step (c) may be for these fragments.
- 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.
- the RFLP is located in a non-coding portion associated with the gene for PrP.
- 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.
- hybridises is used in the context of conditions which produce an informative signal within 70 days (5 ⁇ t1 ⁇ 2 of 32 P ).
- the probe is preferably at least 20 bases long.
- 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.
- the oligonucleotide:ATC CTC TAT TAT GAA TTC TTC TGG AAA ACT 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).
- 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.
- PCR polymerase chain reaction
- the polymerase chain reaction may be used to amplify the DNA sequence which would include the polymorphic (eg EcoRI) site if present.
- 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 :-
- 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.
- a suitable probe e.g. pEA974
- 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.
- 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.
- fig 1 the result of a Southern analysis of Cheviot sheep DNA digested with HindIII ( Figure 1A) or EcoRI ( Figure 1B) and hybridised with 32 P-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).
- 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 NaH 2 PO 4 , 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 32 P dCTP to 10 9 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.
- Negative line Sip P A P A prp A / A
- Negative Line:- 1 is PrP A / A
- Sources of animals a - AFRC/MRC Neuropathogenesis Unit, Edinburgh; b - Redesdale; c - Moredun; d - Dingwall; e - Thornhill; f - St Boswell.
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9023793A GB2235531B (en) | 1988-05-17 | 1990-11-01 | Detection of polymorphism linked to scrapie susceptibility |
DK273990A DK273990A (da) | 1988-05-17 | 1990-11-16 | Paavisning af modtagelighed for sygdom |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8811608.2 | 1988-05-17 | ||
GB888811608A GB8811608D0 (en) | 1988-05-17 | 1988-05-17 | Detecting disease susceptibility |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1989011545A1 true WO1989011545A1 (fr) | 1989-11-30 |
Family
ID=10637006
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1989/000522 WO1989011545A1 (fr) | 1988-05-17 | 1989-05-15 | Detection de sensibilite a l'encephalopathie spongiforme bovine |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0418277A1 (fr) |
AU (1) | AU619308B2 (fr) |
DK (1) | DK273990A (fr) |
GB (2) | GB8811608D0 (fr) |
NZ (1) | NZ229147A (fr) |
WO (1) | WO1989011545A1 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2258867A (en) * | 1991-08-20 | 1993-02-24 | British Tech Group | Oligonucleotides and the use thereof in an assay for encephalopathies |
WO1996027680A1 (fr) * | 1995-03-04 | 1996-09-12 | Boehringer Mannheim Gmbh | Detection d'acides nucleiques specifique aux sequences |
US5618673A (en) * | 1991-08-20 | 1997-04-08 | British Technology Group Limited | Oligonucleotides and their use in an assay for encephalopathies |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9922482D0 (en) * | 1999-09-23 | 1999-11-24 | Meat And Livestock Commission | Method of diagnosis |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1986007464A1 (fr) * | 1985-06-14 | 1986-12-18 | Genetic Systems Corporation | Procedes d'identification d'alleles associes a un risque accru de diabete |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8716367D0 (en) * | 1987-07-10 | 1987-08-19 | Reeders S T | Genetic identification |
-
1988
- 1988-05-17 GB GB888811608A patent/GB8811608D0/en active Pending
-
1989
- 1989-05-15 WO PCT/GB1989/000522 patent/WO1989011545A1/fr not_active Application Discontinuation
- 1989-05-15 EP EP89906155A patent/EP0418277A1/fr not_active Withdrawn
- 1989-05-15 AU AU36957/89A patent/AU619308B2/en not_active Expired
- 1989-05-16 NZ NZ229147A patent/NZ229147A/en unknown
-
1990
- 1990-11-01 GB GB9023793A patent/GB2235531B/en not_active Expired - Lifetime
- 1990-11-16 DK DK273990A patent/DK273990A/da not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1986007464A1 (fr) * | 1985-06-14 | 1986-12-18 | Genetic Systems Corporation | Procedes d'identification d'alleles associes a un risque accru de diabete |
Non-Patent Citations (5)
Title |
---|
Chemical Abstracts, vol. 105, no. 15, October 1986, (Columbus, Ohio, US), G.A. Carlson et al.: "Linkage of prion protein and scrapie incubation time genes", see page 190 * |
Chemical Abstracts, vol. 106, no. 25, June 1987, (Columbus, Ohio, US), R.I. Carp et al.: "Genetic control of scrapie: incubation period and plaque formation in I mice", see page 417 * |
Chemical Abstracts, vol. 108, no. 15, April 1988, (Columbus, Ohio,US), J. Hope et al.: "Molecular pathology of scrapie-associated fibril protein (PrP) in mouse brain affected by the ME7 strain of scrapie", see page 544; * |
Chemical Abstracts, vol. 197, no. 21, November 1987, (Columbus, Ohio, US), N. Hunter et al.: "Linkage of the scraple-associated fibril protein (PrP) gene and Sinc using congenic mice and restriction fragment length polymorphism analysis", see page 205 * |
Proc. Natl. Acad. Sci. USA, vol. 83, September 1986, N.K. Robakis et al.: "Isolation of cDNA clone encoding the leader peptide of prion protein and expression of the homologous gene in various tissues", pages 6377-5381 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2258867A (en) * | 1991-08-20 | 1993-02-24 | British Tech Group | Oligonucleotides and the use thereof in an assay for encephalopathies |
WO1993004198A1 (fr) * | 1991-08-20 | 1993-03-04 | British Technology Group Ltd | Oligonucleotides et leur usage dans la detection des encephalopathies |
GB2258867B (en) * | 1991-08-20 | 1995-05-24 | British Tech Group | Oligonucleotides and their use in an assay for encephalopathies |
US5618673A (en) * | 1991-08-20 | 1997-04-08 | British Technology Group Limited | Oligonucleotides and their use in an assay for encephalopathies |
WO1996027680A1 (fr) * | 1995-03-04 | 1996-09-12 | Boehringer Mannheim Gmbh | Detection d'acides nucleiques specifique aux sequences |
Also Published As
Publication number | Publication date |
---|---|
GB2235531A (en) | 1991-03-06 |
GB9023793D0 (en) | 1990-12-19 |
GB2235531B (en) | 1991-08-07 |
AU619308B2 (en) | 1992-01-23 |
EP0418277A1 (fr) | 1991-03-27 |
GB8811608D0 (en) | 1988-06-22 |
DK273990D0 (da) | 1990-11-16 |
DK273990A (da) | 1990-11-16 |
NZ229147A (en) | 1991-12-23 |
AU3695789A (en) | 1989-12-12 |
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