OLIGONUCLEOTIDES AND THEIR USE IN AN ASSAY FOR ENCEPHALOPATHIES

Abstract: Oligonucleotides of formula: (X)a-(6N-palindrome)n-(Y)b, wherein "6N-palindrome" represents TACGTA, ACGTAT, CGTATA, GTATAC, TATACG or ATACGT (read in the conventional notation with the 5'-end at the left); n is an appropriate integer of at least 2; X is a length of appropriate 5'-end or tailing DNA and a is 0 or 1; and Y is a length of appropriate 3'-end or heading DNA and b is 0 or 1; or any labelled form thereof or any derivative thereof which retains the ability of the 6N-palindrome sequence to anneal or hybridise to its complementary sequence are useful in assay of encephalopathies such as scrapie, BSE or CJD. Preferably they are used as PCR primers to amplify DNA in the sample. PCR product can be detected by restriction with an appropriate enzyme and comparing the restricted and unrestricted products.

Description: OLIGONUCLEOTIDES AND THEIR USE IN AN ASSAY FOR ENCEPHALOPATHIES Background of the Invention 1. Field of the Invention This Invention Is 1n the field of assay of encephalopathles 1n humans and animals. The term "encephalopathy" 1s used herein to refer to neurodegeneratlve diseases Implicating prlons such as scrapie, 1n sheep and goats, bovine sponglform encephalopathy (BSE) 1n cattle, mink and cats and Creutzfeld-Jakob Disease (CJD), Gerstmann-Straϋssler-Schelnker Syndrome (GSS) and kuru 1n humans. The term "assay" 1s used herein to cover mere detection (the Hkely ordinary use envisaged) and a quantitative or semi-quantitative procedure in which some estimate 1s made of the amount of the DNA present 1n the sample. 2- Description of the related art The state of knowledge of the causative agent of encaphalopathles has been summarised recently by N. Hunter, TINS _L4 (9), 389-390 (1991) and by S. B. Prusiner, M. Torcha and D. Westaway, Cornell Veterinarian 8J. (2), 85-101 (1991). The Infectious agent 1s a particle which differs from a virlon and has been termed a "prlon". Prlons are thought to contain little or no nucleic add and are composed largely of protease-reslstant protein (PrP), which 1s encoded by a cellular gene of the host. This feature distinguishes prlons sharply from virlons. To date, no prion-spedf1c nucleic add has been Identified which is required for transmission of disease. Virus-like tubulofilamentous particles 23-26 nm in diameter have been seen consistently In the brains of all known sponglform encephalopathles. These particles have been investigated by H. K. Narang, Intervlrology .32, 185-192 (1991) and H. K. Narang, D. M. Asher and D. C. Gajdusek, Proc. Natl. Acad. Sci. USA 135, 3575-3579 ( 1988 ) and S , 7730-7734 ( 1987 ) . They have a core of prlons 1n a rod-like form. The prlon rods are also termed scraple-asso ated fibrils (SAF). Over the core is a layer of DNA, removable by DNAse and above that lies an outer protein coat which is digestible by a protease. In the Intervirology paper 1t 1s noted that no evidence as yet supports the existence of scrap1e-spedf1c nucleic add, but 1t will be important to purify tubuiofilamentous particles in order to characterise the nucleic acid and determine Its relationship to the disease. Very recently H. . Narang, N. S. Millar, D. M. Asher and D. C. Gajdusek, Intervirology 2Z, 316-324 (1991), have reported an Increase in multimerlc m1tochondr1al DNA 1n the brain of scrapie-lnfected hamsters, but, again, stated that there was no evidence for a scraple-spedfic nucleic acid. In their review entitled "The Search for Scrapie Agent Nucleic Add", J. M. A1ken and R. F. Marsh, Mlcroblol. Rev. 54 (3) 242-246 (1990), concluded that the lack of success in identifying a scraple-spedfic nucleic add suggests that if there 1s such a nucleic acid, 1t would appear to be a very rare, or very small RNA or an RNA or DNA species having significant sequence similarity to nucleic acids present in unlnfected tissue. Similarly, N. Meyer, V. Rosenbaum, B. Schmidt, K. Gilles, C. Miranda, D. Groth, S. B. Prusiner and D. Rlesner, "Search for a putative scrapie genome 1n purified prlon fractions reveals a paucity of nucleic adds", J. Gen. Virol. 11, 37-49 (1991) noted that prlons resist Inactivation by harsh procedures that specifically hydrolyse or modify nucleic adds, a feature which argues that they are probably devoid of polynucleotldes, but that such negative results are always vulnerable to the criticism that a putative scraple-spedfic nucleic add might be so well protected by some unusual structure that its presence 1s yet to be detected. These authors concluded that the nucleic add would have to be very small (of size less than 100 nucleotides), very efficient or heterogeneous in size. The most reliable method, of detecting an encephalopathy is hlstologically, especially by electron microscopy. See H. K. Narang and R. H. Perry, The Lancet, 3___j_, 663-664 (March 17, 1990), and H. . Narang, D. M. Asher and D. C. Gajdusek, Proc. Natl. Acad. Sd. USA , 7730-7734 (1987), but this requires brain tissue and 1s a slow and painstaking method. It would be desirable to have a method of diagnosis based on nucleic add Identification. Such methods have been suggested. See H. Soru , B. Hyllseth, R. rona and 0. Olsvlk, Abstracts of the Annual Meeting of the American Society of M1crobiolog1sts 1988, page 363, Abstract No. C-188, who used a probe of DNA derived from the gene sequence coding for prlon protein. Since, however, 1t 1s well known that prlon protein 1s encoded by a normal chromosomal gene found in all mammals, including those affected by encephalopathles, the above-cited work has not gained acceptance. PCT Patent Application Publication No. W089/11545 (Institute for Animal Health Ltd.) purports to describe a method of detection of scrapie by use of a restriction fragment length polymorphism (RFLP) linked to the so-called Sine gene associated with short Incubation times of sheep infected by scrapie. The RFLP 1s said to be located 1n a non-coding portion associated with the gene for prlons. At best, this method would detect only sheep with the short incubation time characteristic. Hitherto, methods of diagnosis based on nucleic add identification have not been successful or even likely to be successful, since an encephalopathy specific nucleic add has eluded detection despite numerous attempts. Several press articles 1n 1991 1n the English newspaper "The Guardian" (27 April, 22 June, 16 August) report that the present Inventor believes that he has found the genetic fingerprint for BSE and related diseases. No further Information about the nature of the genetic fingerprint is given. Summary of the invention By a remarkable piece of research, the present inventor has found a scraple-spedfic nucleic add, which is also believed to be specific for other encephalopathles. The apparently unique nature of this nucleic add forms the basis of a method of diagnosis which typically Involves either (a) using a sequence from 1t as the basis of a DNA amplification, especially polymerase chain reaction (PCR), primer to make a sufficient amount of it for detection by a restriction fragment length method or (b) labelling 1t and using It as an oligonucleotide probe 1n a sensitive assay which includes a step of hybridising the probe to nucleic add of the sample. The assay can be carried out on body fluids. The scraple-spedfic nucleic acid is single-stranded DNA and includes the sequence (TACGTA) n where n is at least 6. The basic six nucleotide unit of this repeat sequence is pallndromic, 1n the sense that a complementary DNA would have the same TACGTA sequence when read in the 5' to 3' direction. The full length sequence of the DNA 1s not yet known, but it is suspected that n is very much larger than 6, perhaps of the order of 20-30 and further that the DNA 1s probably of the general form:- (TACGTA) n -L (TACGTA) n -L 2 -(TACGTA) n -L 3 .... wherein n-, , n 2 , n 3 ... 1s a series of Integers which may be the same or different and L- | , L 2 , L 3 ... 1s a series of Unking lengths of DNA which may be the same or different. The general form shown might be headed at the 3'-end and/or tailed at the 5'-end by other DNA, of unknown sequence. The number of (TACGTA) n blocks, i.e. the extent of the series n**, n 2 , n 3 .... is not yet known, nor 1s therefore the number of linkers L- j , L 2 , L 3 Nevertheless, it is already apparent that the TACGTA sequence is valuable in diagnosis. A search has shown it to be known in the form TACGTA unrepeated, I.e. n-1 , 1n 93 genes, the double sequence (TACGTA) 2 , I.e. n-2, in certain DrosophHa and yeast genes, but higher repeats, i.e. n-3 or more, are believed unique. The present Invention provides oligonucleotides of formula (X) a -(6N-palindrome) n -(Y) b (1) wherein: "6N-palindrome" represents TACGTA, ACGTAT, CGTATA, GTATAC, TATACG or ATACGT (i.e. the paϋndromlc sequence beginning at any one of the six nucleotldes thereof), n 1s an appropriate Integer of at least 2; X 1s a length of appropriate 5'-end or heading DNA and . 1s 0 (i.e. no heading DNA is present) or 1; and Y Is a length of appropriate 3'-end or tailing DNA and ]_. 1s 0 (I.e. no tailing DNA 1s present) or 1; or any labelled form thereof or derivative thereof which retains the ability of the 6N-pal1ndrome sequence to anneal or hybridise to its complementary sequence). "Appropriate" means compatible with possible use of the oligonucleotide as a DNA amplification, e.g. PCR, primer or in hybridisation, e.g. as a labelled probe. There are, of course, practical limits to the length of the oligonucleotide depending on Its Intended use. For use as a primer n 1s normally 2, 3 or 4. For use as a probe the length will not normally exceed 200 nucleotldes, preferably not more than 70, in all. The term "label" refers to any detectable substance or residue of a substance. The term "oligonucleotide" excludes complete genes or copies of genes or of complete cDNA complementary to complete mRNA transcribed from genes. The invention Includes the above-defined oligonucleotides both fie ie_ and for any use 1n or relating to assay of an encephalopathy. In a third aspect, the invention Includes a kit for carrying out a polymerase chain reaction, the kit comprising, I.e. consisting of or Including, (1) deoxyrlbonucleotides, (2) a polymerase suitable for the PCR and (3 ) at least one oligonucleotide of the Invention as defined above, as a primer. Such kit components are normally provided within .separate containers which together form the kit or part of the kit. Fourthly, the Invention Includes specifically a method of assay for an encephalopathy, which comprises treating a sample suspected of containing DNA specific for the encephalopathy to amplify the DNA, for example by the PCR, detecting the product of the amplification and thereafter analysing the product for the presence of multiple copies of the TACGTA repeat sequence, especially by restricting the product enzymically and comparing the restricted product with unrestricted product. Fifthly, the Invention Includes specifically a method of assay for an encephalopathy which comprises probing a sample suspected of containing DNA specific for an encephalopathy, optimally after amplification of the DNA, with a labelled complementary probe DNA, under appropriate hybridisation conditions and assaying the hybrid product thus obtained. It 1s envisaged that the complementary DNA would normally comprise a ( 6N-palindrome) n sequence, although 1t cannot be excluded that it could alternatively comprise Unking sequence (L- j , L 2 , L 3 ) or some part thereof. Such a linking sequence could be of formula (A)p(TA)q(T)r where p Is 0 or 1 and q is a number from 1 to 20, especially from 3 to 9 and r 1s 0 or 1. Of course, 1t could comprise both types of sequence. Sixthly, the invention further includes scraple-spedfic DNA which comprises at least one sequence of formula (TACGTA) n where n 1s at least 6. It may optionally have other components of sequence, as Indicated above. Description of the preferred embodiments (a) The oligonucleotides Although the invention Includes the oligonucleotides ss∑ ι_, the definition of their preferred features pays attention to their chief presently contemplated use, namely as DNA amplification primers. For this purpose n in formula (1) Is preferably 2, 3 or 4 and the minimum preferred length of palindromic sequence is about 12. The term "pallndromic sequence" herein means any six or more consecutive nucleotldes selected from SEQ ID NO: 1: 1 TACGTATACG TA 12, which can be written as (TACGTA) 2 . In other words, 1t includes palindromlc sequence within "(6N-pal1ndrome)" units and within "(X)" and "(Y)" of formula (1). Less than the preferred minimum of 12 might result 1n an ability to detect DNA having only one TACGTA block, which is not too uncommon, but this might be tolerable In some circumstances, e.g. where more than one detection method Is being used. Even with 12 bases, e.g. (TACGTA) 2 , there 1s some possibility of detecting a twice-repeated sequence 1n a yeast, although there could be further circumstances 1n which this possibility could be well tolerated. A more preferred length of pallndromic sequence is from 12 to 24, specially 16 to 24, and most especially 17 or 18. A palindromlc sequence longer than 24, e.g. (TACGTA^TA, could be tolerable 1n some circumstances, but there would probably be a self annealing problem. That is to say, the 3'-end of the primer could anneal to its own 5'-end instead of to the encephalopathy-spedflc DNA of the sample. Indeed, 1t is this very capacity for self-annealing, scrapie-spedfic DNA, which probably explains why the DNA has evaded detection previously. The oligonucleotides could be modified 1n various ways, starting from the fundamental repeated pallndromic sequence, to serve as primers. Extension of the primer proceeds from the 3'-end. Therefore, primers could have an "irrelevant" 5'-tail, for example of formula (N) m where N 1s any single nucleotlde, e.g. A, C, G or T, and m 1s sufficiently large to create a poly-N tail of very low probability of occurrence in natural sequences, e.g. 8 or more, especially 10 or more. Preferably, however, a 5'-tail consists of or Includes the sequence (A)p(TA)q where j_ Is 0 or 1 and JJ 1s at least 1, especially from 1 to 3, immediately to the 5'-end of (TACGTA) n . There 1s no definite upper limit to the length of the 5'-ta1l, but 1f 1t is too long 1t 1s likely to interfere with the PCR. Tails of moderate length have been previously proposed for PCR primers, principally as a means of Unking them to an Insoluble support, see e.g. U. K. Patent Specification 2233654A (NRDC). Such a tail Is not necessarily composed entirely of nucleic add. Formula (1) above allows for the possibility that the primers will not consist of palindromlc sequence in an exact whole number of repeats, e.g. consist of (TACGTA) n with no other nucleotldes at either end. Thus, the head and/or tall elements can comprise residues of pallndromic sequence, arranged, of course, In the same order as 1n the repeat palindromlc sequence. For example, a 17-mer primer could have the SEQ ID NO: 2: 1 ATACGTATAC GTATACG 17 which 1s of formula (1) wherein "6N-pal1ndrome" is TATACG, n is 2, 1 Is 0, b. Is 1 and the 5'-ta1l Is ATACG. Ordinarily, for use 1n the PCR, 1. will be 0, as It Is not desirable to have any nucleotldes ahead of the 3'-end of the palindromlc sequence, as they would inhibit primer extension. However, the mismatch of a single nucleotide might be tolerable, e.g. 1n the 18-mer SEQ ID NO: 3: 1 TATACGTATA CGTATACN 18 where nucleotide N at the 3'-end is A, C or T and therefore not a residue of the palindrome (G 1s required for the palindrome). When the oligonucleotide 1s Intended to be used in a method of assay Involving a hybridisation step, It can also consist entirely of palindromlc sequence or also contain non-pal1ndromlc nucleotldes to the 5 1 - or 3'- end or both of the palindromlc sequence. Such non-pal1ndromlc sequence could be any unlikely to Interfere with the assay. For example, 1t could consist of or Include a sequence (A)p(TA ) q, as mentioned above, at the 5'-end or, complementarlly, 3' (T)p(AT)q 5' at the 3'-end of a sequence 3' ATGCAT 5'. For use in the PCR reaction the oligonucleotide will not ordinarily be labelled except possibly at Its 5'-end, see e.g. A.-C. Syvanen, M. Bengstrom, J. Tenhunen and H. Soderlund, Nucleic Acids Research 16_, 11327-11338 (1988). In principle, it could be labelled 1n any other way which does not interfere with annealing or primer extension, 1f so desired. For use 1n an assay which Involves hybridisation, the oligonucleotide will normally be used 1n a labelled form, labelling being by any appropriate method such as radiolabelling, e.g. by ^ 2 P, 35 S or by biotinylation (which can be followed by reaction with labelled avldln or streptavidln). However, 1t 1s also possible to use an unlabelled oligonucleotide as a probe provided that 1t Is subsequently linked to a label. For example, the oligonucleotide could be provided with a poly-C tall which could be linked subsequently to labelled poly-G. The oligonucleotides of the invention will not ordlnarly be longer than 200 nucleotldes, even when used as probes, and in practice are likely to be very much shorter, especially up to 70 nucleotldes and more especially up to 45 nucleotldes long. Thus, especially for PCR purposes, they are unlikely to comprise more than 24 nucleotides of the palindrome plus an optional 5'-end or tail of (say) 8-20 nucleotldes, making 32-44 nucleotldes 1n all. (b) The assay method In the preferred method, the oligonucleotide 1s used to amplify the sample DNA. This 1s conveniently performed at present by the PCR, but, 1n principle, any other method could be used which Involves using the oligonucleotide to prime the synthesis of or otherwise create a second strand of DNA using the single-stranded DNA of the sample as a template. The PCR can be applied very easily by using a commercially available kit and an oligonucleotide of the invention as a primer. Because of the repetitive nature of the sequence of the DNA of the sample, the same oligonucleotide can be used as both a forward and reverse primer. Commercial kits normally consist of deoxynucleotides (A, C, G and T), a polymerase which will withstand the temperatures used 1n the PCR such as I&α, polymerase, buffers and control DNA for testing the kit. Therefore, the oligonucleotide primer can be sold on Its own for use with the kit. However, 1t is also possible to sell an add-on kit comprising at least one oligonucleotide primer of the invention, a restriction enzyme for cutting the pallndromic sequence and optionally other components such as a length of control DNA similar to the encephalopathy-spedflc DNA, for testing the add-on kit. The PCR conditions can be any known for use 1n this kind of assay, conveniently those recommended for use with the kit. The PCR will yield a product 1n the form of DNA of varying lengths containing the pallndromic sequence. This can be analysed in any desired way, but a method relying on restriction by an enzyme is probably the easiest. The PCR product will yield bands of various molecular weights. In some Instances the encephalopathy-specific DNA will be primed near Its 3'-end, which will generate multiple copies of large molecules. The PCR product is divided into two portions. The first 1s run on a resolving gel to show a band of high molecular weight associated with the encaphalopathy-spedfic DNA. The second portion is restricted with a restriction enzyme which cuts the pallndromic sequence. This will severely reduce the length of the longer DNA and eliminate certain other bands of shorter DNA altogether. Multiple restrictions of TACGTA will produce many bands of too low a molecular weight to be detected. By comparing the restriction fragment length patterns obtained from the two portions, 1t can readily be determined whether the encaphalopathy-spedfic DNA 1s present in the sample. Examples of suitable enzymes are ϋnaBI and AccI which cut between the C and G of TACGTA and Mael and Snai which cut between the A and T, i.e. between one TACGTA sequence and the next. All these enzymes leave blunt ends and all recognise the six-base sequence. In the alternative, hybridisation, method, the sample DNA could be first amplified, e.g. by a PCR method, or used as 1t is. A hybridisation probe is preferably from 16 to 100 nucleotldes, especially from 16 to 45 long. The hybridisation assay can be carried out in any of the conventional ways. Southern blotting will probably be very convenient. (c) The samples The invention can be applied to assay of samples from any body tissue or fluid in which the Infectious agent and therefore the encephalopathy-spedfic DNA is Hkely to be present 1n a reasonable concentration, especially in whole blood, serum, plasma, cerebrosplnal fluid, saliva, kidney, spleen, liver, and placenta. Brain and spinal cord could be used 1f desired. (d) The encephalopathles Although the Invention has been pioneered using the DNA from the infectious particles of scrapie, current opinion is that BSE, scrapie, CJD and other encephalopathles probably result from the same Infectious agent, transferred to another species. If 1t is not precisely the same, then 1t 1s probably very similar. It is therefore believed that the TACGTA palindromlc sequence appears in al1 the known encephalopathles and possibly others. Accordingly, the sample can be taken from any animal, including those mentioned above, or human believed to be suffering from an encephalopathy. The following Examples illustrate the Invention. EXAMPLE 1 Example 1 describes the isolation and partial sequencing of a scraple-spedfic single-stranded DNA. MATERIALS AND METHODS (a) Animals A total of 200 weanling female Golden Syrian hamsters were inoculated intracerebrally with the 263K strain of scrapie agent using 0.03 ml of a 10 suspension of scrapie-lnfected hamster brain. Clinically ill animals (65-90 days post inoculation) and a similar number of control, age-matched hamsters were killed in batches of 20. b) Purification of nucleic add Isolation of DNA was carried out at 0-4°C (unless otherwise stated) in sterile solutions. The brains of the hamsters were removed and each brain was homogenised 1n 10 ml of 0.32 M sucrose (0-4°C) containing 10 M MgCl 2 1n Griffith tubes. The homogenates were centrifuged at 0-4 β C, at 725 x g for 10 mln. to remove host nuclei. The supernatant at 0-4 β C was further centrifuged at 40,000 x g for 1 hour. The pellet from 20 brains was resuspended in 5 ml of 10 M MgCl 2 in water at 0-4 β C to which rlbonuclease A (RNase) bovine pancreatic type III-A ( Sigma), 20,000 units and deoxyrlbonuclease type 1 (DNase), (Sigma) 20,000 units were added. The mixture was Incubated at 37 β C, mixing every 15 m1n. After Incubating for 1V_ hours with the nucleases TE buffer (20 M Tr1s, 5 mM EDTA, pH 8.5) was added to a final volume to 30 ml (0-4 β C). The mixture was centrifuged (0-4 β C) again at 40,000 x g for 1 hour, the pellet resuspended 1n 5ml Tris EDTA buffer (pH 8.5 at 0-4 β C), 10 mg/ml proteinase K (Boehringer) added and incubated for ]% hours at 42 β C mixing every 15 m1n. Sodium dodecyl sulphate (SDS) was added to give a final concentration of 0.5% and nucleic add was extracted from the mixture using the phenol/chloroform extraction procedure (Sambrook, Frltsch & Maniatis, 1989). The nucleic add was precipitated 1n ethanol and resuspended 1n 25 μl/per brain Tris EDTA buffer pH 7.4 (TE). (c) Further purification of nucleic add 500 μl aϋquots (nucleic add from 20 brains, In ten aϋquots) of the resuspended nucleic add were re-treated and incubated for one hour at 42 β C with 10 mg/ml proteinase K. The nucleic acid was re-extracted from the mixture using the above phenol/chloroform extraction procedure and precipitated in ethanol and resuspended in 500 μl TE buffer. The preparation was re-treated by Incubating with 200 units RNase (heat inactivated for DNase) in 1Q M MgCl 2 buffer for one hour at 37 β C. The nucleic add was re-extracted from the mixture using the above phenol/chloroform extraction procedure and precipitated in ethanol so that each tube contained DNA from five brains. For the alkaline gel, the nucleic add was resuspended 1n 500 mM NaOH and incubated at 65 β C for 90 mln. The DNA samples were loaded containing 6X alkaline loading buffer Into the wells of the alkaline gel. 6X alkaline loading buffer consists of 300 N NaOH, 6 mM EDTA, 18% Ficoll (Type 400; Pharmacia) 1n water, 0.15% bromocresol green and 0.25% xylene cyanol FF. (d) Agarose gel electrophoresls Nucleic add was separated by electrophoresls 1n 1.0% agrose (BRL) on horizontal slabs 1n TAE buffer (40 mM Tr1s-acetate, pH 7.6, 1 mM EDTA). Gels were stained with eth1d1um bromide which was Incorporated 1n the agarose gel at a concentration of 2 μg/ml . Gels were run at 75 mA constant current. Lambda DNA Hindlll digest fragments and 1 kb size markers (BRL) were used. (e) Alkaline agarose gel To prepare alkaline agarose gel a known amount of agarose (BRL) in distilled water was melted and equilibrated at 50°C and 3 M NaOH was added to give a final concentration of 50 mM NaOH, 2 M EDTA. When purification of the band was required from the gel, low melting agarose (BRL) was used. To prepare this gel, first a normal agarose bed was made with 1% agarose and, after the gel had set, the low-melting alkaline gel was poured on top as usual. It would normally be considered ridiculous to add ethidlum bromide to an alkaline agarose gel for resolution of DNA. The ethidlum bromide staining agent works by Intercalating between strands of double-stranded DNA, but on an alkaline gel, double-stranded DNA would become single-stranded. In this Instance, it was added (2 μg/ml) Into the alkaline gel by mistake. Electrophoresls was carried out 1n 50 mM NaOH alkaline electrophoresls buffer at 40 mA constant current as described in detail by M. W. McDonell, M, N. Simon and W. Studier, J. Mol . Blol. 11P_, 119-146, (1977). (f) Purification of nucleic add from the alkaline gel After electrophoresls on alkaline low melting agarose gel, a discrete band of about 1.2 kb of nucleic add remained stained with ethidlum bromide, but only 1n the lane loaded with nucleic acid of the scrapie-lnfected material. This 1.2 kb band which remained stained under alkaline conditions was cut from the gel. The piece of gel was neutralised by several changes in 20 volumes of 1.5 M NaCl and 1 M Tris buffer (pH 7.6). The concentration of NaCl and Tris was gradually dropped to 10 mM each. Finally the gel slice was weighed 1n a 1.5 ml micro-centrifuge tube and 3 volumes of distilled water added. The tube was incubated in a water bath at 60 β C.for 5 min until the agarose had melted. Various unsuccessful attempts were made to further purify the nucleic acid. Best results were obtained without any further purification of this DNA in low melting agarose suspension which was used for synthesis of the second strand of cDNA. Various methods and combinations including the self-priming method by Klenow fragment of £. coll DNA Polymerase, to Hnk synthetic DNA Linkers with terminal transferase were tried. An attempt was also made to Hnk the scraple-spedfic single-stranded DNA by terminal transferase to the plasmld DNA. All these methods used are described In detail 1n "Molecular Cloning, a Laboratory Manual", 2nd ed., J. Sambrook, E. F. Frltsch and T. Man1at1s, Cold Spring Harbor Laboratory Press, 1989. The only method which was successful 1s described below. 10 μl suspension of the purified DNA 1n water from low melting agarose, 1 μl of synthetic random 14-mer oligonucleotide primer NEP-112X (Du Pont) 1n ISJJ Polymerase buffer was heated at 95-100 β C for 5 min. The tube was allowed to cool slowly to 37°C, the resulting condensation was centrifuged and the tubes re-heated and cooled slowly to 37°C twice, (with buffer only). 2 μl of 0.5 mM dNTPs mixture and one unit of AmpHTaq DNA Polymerase (Perkln Elmer Cetus) was added. The mixture was heated for three m1n at 80 β C and then at 75°C for 5 min. An additional unit of AmpHTaq DNA Polymerase was added and temperature was gradually dropped, to 70°C and then to 65 β C, 60°C, 55°C and 50 β C, holding each temperature for 5 min. No PCR was carried out at this stage. The double-stranded DNA synthesised was again purified by phenol/chloroform extraction and ethanol-precipltated. The pellet was resuspended 1n 20 μl of distilled water and treated with 5 units of S- j nuclease (Pharmacia) for 15 min at 37°C and again purified by phenol/ chloroform extraction method and ethanol-precipltated. (g) Construction of a recomblnant plasmid The cDNA synthesised was 11gated into M13mpl0, Sma 1 cut and dephosphorylated (Amersha ), with T4 DNA Ugase. The recomblnant phage was transformed into the competent £. coll strain DH5a (BRL) and plated on LB agar plates Incubated overnight at 37 β C as described in "Ml3 Clon1ng/D1deoxy Sequencing Instruction Manual" (BRL). (h) DNA sequence analysis Single-stranded phage DNA was Isolated from 6 hour 1.5 ml of liquid culture as described In "Ml3 Clon1ng/D1deoxy Sequencing Instruction Manual" (BRL) and was sequenced directly by the dideoxy-nucleotlde method using Klenow Fragment, DNA Polymerase with the Ml3-pr1mer SEQ ID NO: 4: 5'-CCCAGTCACGACGTTGT-3' . Annealing reaction mixture containing 1.5 to 2.0 μg of the single-stranded DNA, 3 μl (5X) of sequencing buffer and 1 μl primer in a total volume of 15 μl was heated at 95-100 β C for 5 min and the mixture was left to cool in the heating block to room temperature about 45 min, and briefly microfuged. To 15 μl annealed/primer mixture, 1 μl 0.1M DTT, 1 μl of 35 S dATP (Amersham) and one unit Klenow Fragment Polymerase (DuPont ) were added and tubes incubated for 3 in at room temperature. This mixture was dispensed Into four tubes containing 2 μl 0.1 mM dNTPs and ddNTPs sequencing mixtures. The tubes were Incubated for 30 m1n at 37°C, then briefly microfuged and 2 μl of 0.4 M of sequence mixture, without ddNTPs, but which also contained unlabelled dATP, was added to each tube. After a further 20 min incubation at 37 β C, 5 μl of stop solution was added to each tube and mixed by centrlfugation. Samples were run In 6% acrylamide gel containing 7M Urea 1n Tris-borate electrophoresls (TBE) buffer at 60 watts (40mA). Samples were boiled for 5 m n and cooled on wet Ice before loading. One sample from a clone designated "Nar 50" was also sequenced by the automated processes of the Model 370A DNA sequencer (Applied Bios) at University of Durham (England) with dye Primer C-21M13]. Nucleotide sequencing data was compared with the GenBank database by a computer program. (i) Amplification of clones by the PCR Clones of M13 double stranded DNA and single-stranded DNA with the insert were used for amplification by polymerase reaction (PCR) with -20 and reverse primer of M3. Reactions were carried out 1n a 100 μl reaction mixture 1n a Perkin Elmer Cetus PCR kit according to manufacturer's Instructions. Forty cycles of PCR were carried out on all samples as follows: denaturatlon for 60 s at 94 β C, annealing of primers for 150 s at 45 β C and extension for 120 s at 75 β C. PCR products were analysed on 1.0% agarose gel with ethidlum bromide staining. RESULTS Analysis of nucleic add fraction of both scrapie-lnfected brain and the equivalent fraction of unlnfected brain Isolated and run 1n neutral agarose gel with TAE buffer revealed significant differences between scrapie-lnfected and unlnfected samples, as described 1n detail by H. K. Narang, N. S. Millar, D. M. Asher and D. C. Gajdusek, Intervirology 1991, 21, 316-324 (1991). Briefly, 1n both samples a band was present corresponding In size to that of hamster mltochondrial ( mt ) DNA, a double stranded circular molecule of approximately 15.7 kb. In addition to this band, several broad bands of high molecular weight were seen only in the scrapie-lnfected sample. In the said previous study 1t was shown that the slower migrating bands of nucleic acid were multimeric forms of circular mtDNA. Analysis of nucleic add fraction of scrapie-lnfected brain and the equivalent fraction of unlnfected brain, isolated and run in the alkaline agarose gel with NaOH stained with ethidlum bromide, revealed that one band of about 1.2 kb remained visible under the alkaline conditions 1n the lane with scrapie-lnfected brain, while no DNA markers were seen (these having been denatured under the alkaline conditions). All these nucleic add bands were visualised after the gel was neutralised with NaCl and Tris buffer. The 1.2 kb scrapie-spedfic band was excised, and synthesised into double strand of cDNA as described 1n Methods. Three clones with Inserts at the ≤πιa.1 cut site were Investigated. One had an Insert of only 13 bases, SEQ ID NO: 5: 1 ATATATATAC GTA 13. The other two sequences read (TACGTA) n TA and both were difficult to read after 38 bases. One of the two clones "Nar 50" (with 38 base reading) was also sequenced by the automated system and results were found to be the same. The reading sequence of six bases TACGTA was repeated over and again 1n the same order. There is a restriction site for SnaBl cutting TACGTA 1n the middle. There is also a single site in Ml3 for SnaBl restriction enzyme. The double stranded DNA of Ml3 plasmid with the Insert was prepared from £. coli by a small-scale preparation method. The DNA was cut with the SJQUBI restriction enzyme. Gel analysis revealed two bands one containing over 5,000 base and the other about 2,250 bases. Because of the large size of the two fragments produced, it was difficult to estimate the size of the insert in them. Analysis of 10 μl of the amplified PCR product of Ml3 double stranded DNA and single-stranded DNA run 1n the neutral agarose gel with TAE buffer revealed amplification of a single band of about 250 bases. By calculating the bases of M13 Including the primers at about 100 bases, an insert of size of about 150 bases was indicated. The PCR product was cut with EcoRl . Hlndlll. SnaBl or a combination of two enzymes or all three. EcoRl or Hindlll made very little difference to the mobility of the band but SnaBl by Itself or 1n combination with the other two restriction enzymes reduced Its size by about half to 125 bases. Comparison of the nucleotide sequence of the Inserted DNA to the GenBank nucleotide database revealed no significant homology. When repeating the preparative procedure of Example 1, a further sequence, ATAATA, preceding the palindromlc sequence TAGCTA, was detected. This 1s believed to be at least part of an Intermediate (linker) sequence connecting pallndromic sequences. EXAMPLE 2 This Example illustrates a method of diagnostic assay using the PCR. The scrapie etc. level 1n the blood would be 10 3 doses of infectlvity per gram (or cm. 3 ) of blood. (A dose of 1nfectiv1ty Is that which when diluted and Inoculated into mice gives diseases 1n 50% of the mice). This level 1s about constant in the blood. As little as 1 microlitre of blood is Hkely to contain one molecule of the DNA, which is all that 1s needed for the PCR, but in practice one would use a much larger sample. The blood Is treated with proteinase K to remove unwanted proteins from association with the DNA and heated to destroy proteinase K at 95 β C for 10 minutes. The PCR 1s performed using a (TACGTA) 3 primer, mixed nucleotldes In Is_ς polymerase for 40 cycles. The PCR product 1s then run on a gel 1n 10 μl samples. The first cycle of the three steps of annealing with primer, extension and denaturation 1s carried out at 95 β C throughout. Thereafter 40 cycles are carried out at 45-50 β C, 72 β C and 92°C 1n the three steps. One sample of PCR product 1s digested with the restriction enzyme SnaBl. another is left untreated. Where the band due to the PCR product disappears on treatment with the enzyme, the scraple-spedfic DNA 1s present. (The effect of SnaBl 1s to cut the TACGTA sequence, forming many small pieces of DNA). The following Sequence Listing has been prepared by use of the "Patentin" program supplied by the United States Patent Office. The program contains an error which causes nucleotide lengths to be expressed 1n "base pairs" even when the nucleic acid is single-stranded, as here. For "base pairs" read "nucleotldes". SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: NARANG, HARASH K BRITISH TECHNOLOGY GROUP LTD, (ϋ) TITLE OF INVENTION: OLIGONUCLEOTIDES AND THEIR USE IN AN ASSAY FOR ENCEPHALOPATHIES (iϋ) NUMBER OF SEQUENCES: 5 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: MR. R. KEITH PERCY MA CPA, (B) STREET: BRITISH TECHNOLOGY GROUP LTD., 101 NEWINGTON CAUSEWAY, (C) CITY: LONDON, (E) COUNTRY: GB (F) ZIP: SE1 6BU (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: WO Not yet known (B) FILING DATE: (C) CLASSIFICATION: C07 H 21/00 ? C12 N 15/11 ? G01 N 33/53 ? (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: PERCY, RICHARD K (B) REGISTRATION NUMBER: UK PATENT ATTORNEY (C) REFERENCE/DOCKET NUMBER: PF 134648 PCT/RKP (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: +4471 403 6666 X2311 (B) TELEFAX: +4471 403 7586 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Ala Gly Pro Phe Xaa Leu Tyr Xaa Val Asn 1 5 10 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: satellite (B) LOCATION: 6..12 (D) OTHER INFORMATION: /rpt_type= •■ tandem" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ATACGTATAC GTATACG 17 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO ( ix ) FEATURE : (A) NAME/KEY: satellite (B) LOCATION: 1..6 ( D) O THER INFORMATION: /rpt_type= "tandem" (X i) SEQUENCE DESCRIPTION: SEQ ID NO:3: TATACGTATA CGTATACN 18 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (Vi) ORIGINAL SOURCE: (A) ORGANISM: Ml3 primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCCAGTCACG ACGTTGT 17 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATATATATAC GTA 13

Claims: CLAIMS 1. Oligonucleotides of formula (X) a -(6N-pal1ndrome) n -(Y) D (1) wherein: "6N-pal1ndrome" represents TACGTA, ACGTAT, CGTATA, GTATAC, TATACG or ATACGT (read in the conventional notation with the 5'-end at the left); n Is an appropriate Integer of at least 2; X 1s a length of appropriate 5'-end or tailing DNA and a 1s 0 or 1; and Y 1s a length of appropriate 3'-end or heading DNA and _2 1s 0 or 1; or any labelled form thereof or any derivative thereof which retains the ability of the 6N-pal1ndrome sequence to anneal or hybridise to Its complementary sequence. 2. Oligonucleotides according to claim 1 wherein n 1s 2, 3 or 4. 3. Oligonucleotides according to claim 2, wherein: 1 1s 0 or X represents the residue of a 6N-pal1ndrome sequence and a is 1; t Is 0 or Y represents the residue of a 6N-pal1ndrome sequence and Ji is 1, and the total length of pallndromic sequence within the oligonucleotide 1s from 12 to 24. 4. Oligonucleotides according to claim 3 wherein the total length of pallndromic sequence within the oligonucleotide 1s from 16 to 24. 5. Oligonucleotides according to claim 4 wherein "6N-pa11ndrome" represents TACGTA, a 1s 1 and X comprises a sequence of formula (A)p(TA)q where p. 1s 0 or 1 and a 1s at least 1 immediately to the 5'-end of the "6N-pal1ndrome" sequence. 6. Oligonucleotides according to claim 1, 2, 3, 4 or 5 of length not exceeding 200 nucleotldes. 7. Oligonucleotides according to claim 1 , 2, 3, 4, 5 or 6 in a labelled form wherein the label is attached to the 5'-end. 8. Use of an oligonucleotide according to claim 1, 2, 3, 4, 5, 6 or 7, 1n assay of an encephalopathy. 9. Use of an oligonucleotide according to claim 1, 2, 3, 4, 5, 6 or 7, as a primer in DNA amplification. 10. Use of an oligonucleotide according to claim 1, 2, 3, 4, 5, 6 or 7, in a labelled form, as a probe. 11. A kit for carrying out a polymerase chain reaction, the kit comprising: (1) deoxyrlbonucleotides; (2) a polymerase suitable for the polymerase chain reaction; and (3) at least one oligonucleotide according to claim 1, 2, 3, 4, 5, 6 or 7 for use as a primer. 12. A kit according to claim 11 wherein component (3) comprises forward and reverse primers of a said oligonucleotide. 13. A kit according to claim 11 or 12 wherein the oligonucleotide 1s unlabelled. 14. A kit according to claim 11, 12 or 13 which further comprises a restriction enzyme capable of restricting the 6N-pal1ndrome. 15. A kit according to claim 14 wherein the restriction enzyme is SnaBl. Accl. Ma_e_I or £n_al. 16. A method of assay for an encaphalopathy, which comprises treating a sample suspected of containing DNA specific for the encephalopathy to amplify the DNA, detecting the product of the amplification and thereafter analysing the product for the presence of multiple copies of the TACGTA repeat sequence. 17. A method according to claim 16 wherein the DNA is amplified by a polymerase chain reaction. 18. A method according to claim 16 or 17 wherein the product of the amplification 1s restricted enzymically and the restricted product Is compared with the unrestricted product, whereby disappearance of longer lengths of DNA upon restriction Indicates presence of the encephalopathy-spedfic DNA. 19. A method according to claim 18, wherein the restriction enzyme 1s SnaBl, AccI, Mae.1 or Snal• 20. A method of assay for an encaphalopathy which comprises probing a sample suspected of containing DNA specific for an encephalopathy, with a labelled complementary probe DNA under appropriate hybridisation conditions and assaying the hybrid product thus obtained. 21. Scrapie-spedflc DNA which comprises at least one sequence of formula (TACGTA) n where n 1s at least 6. 22. DNA according to claim 21 which comprises at least one sequence of formula A(TA) 3 (TACGTA) n .

Inventors: NARANG HARASH KUMAR, NARANG, HARASH, KUMAR
Applicants: BRITISH TECH GROUP, BRITISH TECHNOLOGY GROUP LTD
Application year: 1992
Main classification: C12Q1
IPC codes: C07H21, C12N15, C12Q1