`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 4 :
`
`
`
`C12Q 1/68, G01N 33/546
`// G01N 33/548
`
`
`
`
`(11) International Publication Number:
`
`W0 86/ 03782
`
`3 July 1986 (03.07.86)
`
`(43) International Publication Date:
`
`(21) International Application Number:
`
`PCT/GB85/00591
`
`(22) International Filing Date:
`
`19 December 1985 (19.12.85)
`
`(31) Priority Application Number:
`
`8432118
`
`(32) Priority Date:
`
`19 December 1984 (19.12.84)
`
`(33) Priority Country:
`
`GB
`
`(71)(72) Applicants and Inventors: MALCOLM, Alan, Dav-
`id, Blair [GB/GB]; 22 Elm Crescent, Ealing, London
`W5 3JW (GB). LANGDALE, Jane, Alison [GB/GB];
`10A Somerset Road, Ealing, London W13 (GB).
`
`(74) Agents: LAMBERT, Hugh, Richmond et al.; D. Young
`& Co., 10 Staple Inn, London WC1V 7RD (GB).
`
`(81) Designated States: AT (European patent), AU, BE (Eu—
`ropean patent), BR, CH (European patent), DE (Eu-
`ropean patent), DK, FI, FR (European patent), GB,
`GB (European patent), HU, IT (European patent),
`JP, KP, LU (European patent), NL (European pa—
`tent), NO, SE (European patent), US.
`
`Published
`With international search report.
`Before the expiration of the time limitfor amending the
`claims and to be republished in the event of the receipt
`of amendments.
`
`(54) Title: IMPROVED SANDWICH HYBRIDISATION TECHNIQUE FOR THE DETECTION OF NUCLEOTIDE
`SEQUENCES
`
`(57) Abstract
`
`An improved sandwich hybridisation diagnostic method in which there is used as the immobilised polynucleotide, a
`single-stranded polynucleotide sequence covalently coupled to particles or beads of a synthetic resin, preferably a cross-
`linked macroporous cellulosic resin. Also disclosed is a particular application of the method to the detection of the abnor-
`mal human (sickle cell) B—globin gene and diagnostic kits for use therein.
`
`
`
`
`
`
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages ofpamphlets publishing international appli-
`cations under the PCT.
`
`Mali x.
`
`AT Austria
`AU Australia
`BB
`Barbados
`BE
`Belgium
`BG Bulgaria
`BR
`Brazil
`CF
`Central African Republic
`CG Congo
`CH Switzerland
`CM Cameroon
`DE Germany, Federal Republic of
`DK Denmark
`Fl
`Finland
`FR
`France
`
`Gabon
`United Kingdom
`Hungary
`Italy
`Japan
`Democratic People’s Republic
`of Korea
`Republic of Korea
`Liechtenstein
`Sri Lanka
`Luxembourg
`Monaco
`Madagascar
`
`MR Mauritania
`MW Malawi
`NL Netherlands
`N0 Norway
`R0 Romania
`SD Sudan
`SE
`Sweden
`SN Senegal
`SU
`Soviet Union
`TD Chad
`TG Togo
`US United States of America
`
`
`
`WO 86/03782
`
`PCT/G385/0059l
`
`IMPROVED SANDWICH HYBRIDISATION TECHNIQUE
`
`FOR THE DETECTION OF NUCLEOTIDE SEGUENCES
`
`FIELD OF' INVENTION
`
`This invention relates to the detection of nucleotide sequences,
`
`in-
`
`cluding both DNA and RNA sequences.
`
`BACKGROUND AND PRIOR ART
`
`The ability to detect the presence (or absence) of a given RNA or DNA
`
`sequence in a sample, e.g. a clinically obtained sample or specimen,
`
`is
`
`potentially of great benefit to mankind. Existing methods of detection are,
`however, extremely'time consuming and labour intensive and are therefore
`
`for use on anything other than a very limited scale.
`
`In many cases also sensi-
`
`In one commonly used procedure, for example,
`tivity is undesirably low.
`specific DNA sequences are detected in complex mixtures by digesting the
`
`sample with an appropriate restriction endonuclease, followed by size frac-
`
`tionation by gel electrophoresis, transfer of the sized fractions from the gel
`
`to a nitrocellulose or other suitable membrane, hybridisation to a radio-
`actively labelled probe, usually a 3ZP-labelled probe, and finally auto-
`
`radiography. Such a procedure is inherently cumbersome and unsuited for
`
`automation. Also, very often the quantity of DNA or other nucleotide
`sequence to be detected and which is transferred onto the membrane is so
`
`small that in order to obtain a detectable signal after hybridisation a highly
`
`radioactive probe has to be used, which is disadvantageous both from the
`
`handling point of view and from the point of view that the radioactive label
`of choice, i.e. 32P, has a relatively short half-life making it unsuitable for
`
`use as a stock reagent.
`
`An alternative to the above is the sandwich hybridisation technique
`
`disclosed by Dunn AR. and Hassell J.A. in (1977) Cell I; 23-26 in which the
`
`sample containing the nucleotide sequence to be detected is contacted with a
`
`complementary fragment immobilised on a membrane, e.g. a nitrocellulose
`
`filter, and to which the sequence to be detected hybridises in a first
`
`hybridisation step leaving an unhybridised tail available for a second hybrid-
`
`isation step with a labelled probe.
`
`In PIP-0079139 a proposal
`
`is made for
`
`the utilisation of
`
`the above
`
`described sandwich hybridisation technique in the identification of micro-
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`wo 86/03782
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`organisms in a sample.
`
`In accordance with that proposal a sample containing
`
`a single-stranded nucleic acid sequence from the micro-organism to be
`
`detected is contacted with two single-stranded fragments obtained from the
`
`genome of the micro-organism in question either directly or by recombinant
`
`5
`
`DNA technology and complementary to different positions of the sequence to
`
`be detected, but not
`
`to each other, the one being immobilised on a solid
`
`carrier, preferably a nitrocellulose filter, and the other being labelled.
`
`Although labelling with labels other than radioactive labels is implied, only
`
`radioactive labelling is disclosed, and specifically labelling with 1251.
`
`In the
`
`10
`
`presence of the single-stranded nucleic acid sequence to be detected, which
`
`hybridises with both the immobilised fragment and the labelled fragment, the
`
`labelled fragment becomes bound to the carrier and can be detected thereon
`
`by autoradiography, thus giving rise to positive identification of the micro-
`
`organism from which that single—stranded sequence originated.
`
`,
`
`15
`
`Whilst the sandwich hybridisation technique provides advantages over
`
`the first described procedure inherent disadvantages remain arising particu-
`
`larly from the finite and quite limited quantity of nucleic acid which can be
`
`immobilised onto the nitrocellulose filter or other membrane.
`
`This
`
`in
`
`particular places limits on both the speed and sensitivity of the detection
`
`20
`
`method. Also it appears that the efficiency of the hybridisation particularly
`
`in the second stage is very low, thus reducing the sensitivity of the method
`
`still further.
`
`In EP-DO70687 a hybridisation diagnostic method is disclosed which uses
`
`light-labelled single-stranded polynucleotide reagents for hybridising with
`
`25
`
`immobilised sample single-stranded polynucleotides.
`
`A variety of solid
`
`supports are suggested for
`
`the immobilised sample single-stranded poly-
`
`nucleotide including activated glass beads, polyacrylamide, agarose or sepha-
`
`dex beads, and cellulose. Various known methods are also suggested for
`
`30
`
`coupling the sample polynucleotide to the support, largely by reference to
`published literature, e.g. Methods in Enzymology Vol. XXXIV, Part B, 463-
`475, 1974 and Vol. XLIV, 859-886, 1976, but no specific examples of an
`
`operative method are given.
`
`It
`
`is also suggested that the sample single-
`
`stranded polynucleotide can be immobilised by contacting the sample under
`
`hybridisation conditions with an immobilised first single-stranded poly-
`
`35
`
`nucleotide reagent complementary to a different and mutually exclusive
`
`portion of
`
`the sample rpolynucleotide to that which is required for
`
`the
`
`
`
`WO 86/03782
`
`PCT/GB85/00591
`
`hybridisation of
`
`the light-labelled polynucleotide reagent, Le. a so-called
`
`sandwich hybridisation procedure. Again no specific examples are given of
`
`the procedure, or of the method by which the first polynucleotide reagent is
`
`attached to the support in the first place.
`
`SUMMARY OF THE INVENTION
`
`In accordance with one aspect of
`
`the present
`
`invention we have
`
`discovered that both the speed and efficiency of the sandwich hybridisation
`
`reaction, particularly the second stage, can be substantially increased by
`
`using solid particles or beads of a macroporous resin as the carrier for the
`immobilised nucleic acid fragment thus providing a method for the detection
`
`of nucleic acid fragments which is capable of providing a much improved
`
`sensitivity;
`
`In particular the use of particles or beads of a cross-linked
`
`macroporous resin such as sephacryl appears
`
`to result
`
`in substantially
`
`improved covalent bonding of the polynucleotide reagent to the support.
`
`In a second aspect of this invention, a particularly sensitive method has
`
`been developed for the detection of the abnormal human (sickle cell) 8 -globin
`
`gene by the sandwich hybridisation of a Dde I digest of the abnormal gene
`
`with an immobilised polyvhucleotide reagent covalently bonded to solid resin
`
`particles or beads, and a second labelled polynucleotide reagent,
`
`such
`
`reagents comprising polynucleotide sequences obtained by restriction endo-
`
`nuclease treatment of
`
`the normal gene and complementary to different
`
`portions of a restriction fragment contained within the digest and containing
`
`the single base change in the sixth codon of
`
`the B-globin gene that
`
`is
`
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`25
`
`characteristic of the abnormal (sickle cell) B—globin gene.
`
`In accordance with the present invention therefore, there is provided a
`
`method for the detection of a given nucleic acid sequence in a sample, which
`
`comprises contacting the sample under hybridisation conditions with a first
`
`reagent comprising solid particles or beads having immobilised thereon a
`
`single-stranded nucleic acid fragment comprising a sequence complementary
`
`to a portion of
`
`the sequence to be detected and with a second reagent
`
`comprising a labelled single-stranded nucleic acid fragment comprising a
`
`sequence complementary to a different portion of
`
`the sequence to be
`
`detected, but non-complementary to the immobilised fragment,
`
`thereby
`
`forming a hybridisation duplex comprising the sequence to be detected
`
`hybridised with both reagents, separating the hybridisation duplex from any
`
`30
`
`35
`
`
`
`wo 86/03782
`
`PCT/GBSS/0059l
`
`remaining unbound label, and detecting the presence, if any, of label bound to
`the hybridisation duplex.
`'
`
`DETAILED DESCRIPTION
`
`this invention preferably
`The first reagent used in the method of
`comprises a single-stranded nucleic acid fragment immobilised onto the solid
`particles or beads by covalent bonding between reactive, e.g. amino, groups
`in the nucleic acid fragment, and reactive groups in or on the solid particle.
`To this end the preferred solid particles used in the method of this invention
`
`are polymer beads formed from a natural or synthetic polymer having
`reactive, e.g. amino or hydroxy, groups attached to the polymer chain. Most
`preferred are cellulosic polymer beads such as those sold under the trade
`
`names Sephadex and Cellex and more particularly macroporous cellulosic
`materials such as Sepharose and Sephacryl.
`Various methods exist and are known for the covalent bonding of
`nucleic acid fragments to such polymer beads, including coupling with carbo-
`diimides via terminal phosphate groups and coupling via cyanogen bromide.
`Particular mention may be made, however, of coupling via diazotisation of
`aromatic amino groups attached to the polymer matrix, which techniqUe is
`disclosed in detail in (1982) Nucleic Acid Res. 19, 22, 1799-1810 and 7163-
`7196.
`
`Preferably the polymer beads will have a particle size in the range 5 to
`50 microns, more preferably 10 to 20 microns.
`
`the labelling of
`Likewise various methods exist and are known for
`single-stranded nucleic acid fragments to provide the second reagent used in
`the method of this invention. Labelling with radioactive isotopes such as 32P
`and 125P has already been mentioned and can be used in the present
`invention. However, it is envisaged that, because of the greater sensitivity
`that can be achieved by the present invention, less highly radioactive isotopes
`can be used as the label and in particular tritium. Also it is envisaged that
`non-radioactive labels e.g. enzymatic,
`fluorescent and chemiluminescent
`
`labels will be suitable, and in many cases may be preferred.
`
`Once the reagents have been prepared, and it is envisaged that these
`will be available in kit form, the method of the invention is extremely simple
`to carry out and is readily adaptable to automatic or semi-automatic equip-
`ment. Essentially it is necessary merely to mix the sample with the two
`
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`
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`
`
`WO 86/03782
`
`PCT/(3385/00591
`
`5
`
`reagents, either simultaneously or successively, under conditions effective to
`
`permit
`
`formation of
`
`the hybridised duplex comprising the two reagents
`
`hybridised to the restriction fragment which is to be detected. The hybridis-
`ation is rapid, and since in contrast to the sandwich hybridisation technique
`of EP-0079139, both the first (immobilised) reagent and the second (labelled)
`reagent can be used to infinite excess, merely by increasing the quantity
`used,
`the amount of bound label can be maximised thereby to provide a
`maximum signal for subsequent detection. Thus the method of the present
`
`invention can be of great sensitivity,
`
`this being determined not by the
`
`10
`
`concentration of the reagents, which effectively can be infinite, but by the
`
`15
`
`20
`
`25
`
`concentration of the sequence to be detected in the sample.
`
`This is
`
`in
`
`marked contrast to the method of EP-0079139 where the concentration of the
`
`immobilised reagent is finite, and relatively low, so that the quantity of label
`bound to the duplex is equally low and is determined not by the concentration
`in the sample of the sequence to be detected, which is usually far in excess of
`
`the quantity of
`
`the immobilised reagent, but by the quantity of
`
`the
`
`immobilised agent.
`
`In the method of the present invention a far higher pro-
`
`portion of the sequence to be detected present in the sample is hybridised and
`
`consequently labelled. Not only that, but as already indicated, there is a
`substantial and surprising increase in the efficiency of
`the hybridisation
`
`reaction using beads as the carrier as opposed to a filter or membrane, e.g. of
`cellulose nitrate.
`
`The sample used in the method of this invention can be a purified
`and/or fractionated sample containing the polynucleotide fragment or frag-
`ments to be identified in single-stranded form, but it is a major advantage of
`this invention that the sequence to be detected does not have to be separated
`
`from the sample prior to labelling as in the Southern blotting technique.
`
`It is
`
`therefore possible in accordance with the present invention to detect a given
`
`polynucleotide sequence in a crude mixture of polynucleotide fragments
`
`30
`
`obtained by the digestion of the original sample, e.g. a clinical sample
`
`containing DNA or RNA, with an appropriate restriction endonuclease and
`
`subsequent denaturation to reduce the restriction fragments to single-
`stranded form.
`
`Following hybridisation the polymer beads or other particles carrying
`the labelled hybridisation duplex are separated, e.g. by centrifuging and
`
`35
`
`washing, from unbound excess label and the presence of bound label detected
`
`
`
`wo 86/03782
`
`,
`
`PCT/GBSS/00591
`
`in the appropriate manner, e.g. by a scintillation counter in the case of a
`radioactive label, or by enzymatic activity in the case of an enzyme label, or
`light detecting means in the case of a fluorescent or chemiluminescent label.
`The method of the present invention has a wide range of applications
`
`arising from the ability rapidly to identify particular nucleic acid (DNA or
`RNA) sequences in a sample. Thus it can be used to identify pathogenic
`micro-organisms in a sample, genetic abnormalities, etc. as well as simply a
`gene mapping exercise. A particular application given by way of example is
`in the identification of sickle cell disease.
`Sickle cell disease is caused by a single base mutation (A + T) in the
`sixth codon of the human B-globin gene. This change from GAG to GTG in
`
`the sixth codon, besides altering the properties of the resultant haemoglobin
`
`coded for by the gene with valine being incorporated into the protein rather
`than glutamine, also results in the loss of the Dde i and Mat 11 recognition
`sites at the 5' end of the B-globin gene. This is diagramatically illustrated in
`
`the accompanying drawing which is a restriction map of Dde I and Hinf I sites
`at the 5‘ end of the B-globin gene. Dde l digestion of abnormal (sickle cell)
`B-globin gene will therefore produce a single 381 bp fragment comprising
`both Fragments B and C in a single continuous length, as opposed to two
`separate fragments, one .of 201 bp (Fragment B) and 180 bp (Fragment C),
`that will be produced by Dde I digestion of the normal B-globin gene. Using
`the techniques already described to immobilise one of Fragment B or
`Fragment C onto polymer beads and to label
`the other,
`two reagents are
`provided capable of rapidly detecting the presence of abnormal sickle cell.
`gene in a sample. Thus digestion of normal B-globin gene with Dde I will
`produce two fragments neither of which is capable of forming a "sandwich"
`with the two reagents. On the other hand, digestion of abnormal (sickle cell).
`B-globin gene with Dde I will produce a single fragment which is capable of
`forming a "sandwich" with the two reagents. When the Dde I digest
`is
`contacted with the two reagents under hybridisation conditions (i.e. after de-
`naturation of the digest) the abnormal (sickle cell) B-globin gene will result in
`binding of the label, and hence a positive signal, whereas the normal B-globin
`
`gene will not.
`In a practical experiment, the above procedure has been mimicked by
`digesting plasmid BFS with Hinf I. Plasmid BFS contains the 1.9 kb Balm Hl
`restrictioh fragment of the human B-globin gene cloned into the single Bam
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`HI site of the vector pAT 153. Digestion of the plasmid BFS with Hinf I
`
`produced a 341 kb fragment (Fragment A) which was recovered from the
`
`product of digestion by electrophoresis and eiectroelution.
`
`After denaturation the 341 kb fragment was immobilised onto macro—
`
`5
`
`porous cellulosic polymer beads (Sephacryl 5500) using the diazotisation
`
`method described by Seed B. in (1982) Nucleic Acids Res. 11;, 1799-1810.
`In a second operation plasmid BFS was digested with Met 11 to produce a
`
`201 bp fragment (Fragment B, see Figure 1) which was likewise recovered
`
`from the digest. Fragment B was then labelled with 32P by nick translation
`
`10
`
`using the technique of Rigby et al. (1977) J. Mol. Biol. 1__13, 237- 251, to a
`specific activity of 1.5 x 107 cpm pg'l.
`
`In two further, operations plasmid BFS was digested with Dde I and Barn
`
`HI. Digestion with Dde I produces short fragments not capable of "sandwich"
`
`hybridisation with Fragments A and B, whereas digestion with Barn HI
`
`15
`
`produces one long fragment which is capable of "sandwich" hybridisation.
`
`In
`
`each case digestion was followed by denaturation to provide single-stranded
`DNA fragments.
`'
`
`Prior to sandwich hybridisation by the technique of the present
`
`in-
`
`20
`
`vention the immobilised Fragment A was prehybridised overnight at 65°C in l
`ml. 40 mM PIPES pH 6.5, 1 mM EDTA 0.6 M NaCl, 0.1% SDS and 250 pg ml'l
`sonicated denatured salmon sperm DNA. Following prehybridisation, sand-
`wich hybridisation was carried out by mixing either the denatured Dde I or
`
`Bam HI BFS digests with immobilised 341 bp Fragment A and labelled 201 bp
`
`Fragment B in 50 pl of the same buffer and allowing the mixture to stand
`
`25
`
`overnight.
`
`The beads were then separated, washed three times for 10
`
`minutes each at 65° with 1 ml of the same buffer but without the salmon
`
`sperm DNA and then counted. The results are shown in Table 1. These
`
`clearly show that with amounts of plasmid BFS of 100 attomoles or over, a
`
`clear distinction can be drawn between the Bam HI digest and the Dde I
`
`30
`
`digest.
`
`In the figures given in Table 1 the low background count obtained
`
`when either the "sandwich filling" was omitted or replaced by sonicated calf
`
`thymus DNA has not been subtracted.
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`8
`
`
`Table 1
`
`
`
`Amount of restricted
`
`cpm on resin
`
`cpm on resin
`
`BFS plasmid
`
`when enzyme is when enzyme is
`
`(attomoles)
`
`Bam HI
`
`Dde I
`
`10,000
`
`5,000
`
`2,000
`
`1,000
`
`500
`
`100
`
`80
`
`60
`
`40
`
`20
`
`552
`
`748
`
`482
`
`266
`
`196
`
`107
`
`90
`
`82
`
`62
`
`75
`
`63
`
`70
`
`65
`
`71
`
`44
`
`62
`
`61
`
`60
`
`57
`
`60
`
`In a further practical experiment,
`
`the above sandwich hybridisation
`
`using the immobilised 341 bp Fragment A and the labelled 201 bp Fragment B
`
`has'been repeated using a Dde I digest of 60 attomoles of human DNA both
`
`from a normal patient and from a homozygote sickle patient. The results
`
`were unequivocal. After background substraction, a typical experiment gave
`
`441 counts 7(10 minutes) for sickle DNA and 153 counts for normal DNA
`
`illustrating and confirming the effectiveness of the method of the invention
`
`in identifying the presence of sickle DNA.
`
`By mixing the Barn HI and Dde I digests of BFS it is possible also to
`
`mimic the case of the sickle heterozygote.
`
`In such an experiment, counts (10
`
`minutes) were obtained using 100 attomoles plasmid DNA as follows: normal
`
`128; heterozygote 296; homozygote 745; thus indicating that the method can
`
`be quantitated to identify heterozygotes as well as homozygotes.
`
`Im summary,
`
`the method of the present
`
`invention has the following
`
`advantages over present technology:
`
`1.
`
`It
`
`is quicker. Electrophoresis, blotting and autoradiography are elimi-
`
`nated. The sandwich hybridisation can be carried out in as little as 8
`
`10
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`WO 86/03782
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`hours;
`
`the separation of beads by gravity, centrifuge or magnetically
`
`takes seconds; and quantitation in a scintillation counter takes a few
`
`minutes.
`
`In practice results have been obtained within 48 hours of
`
`taking blood and the indications are that substantial reductions will be
`possible in the future.
`
`It is extremely easy to automate and hence to process a large number
`of samples.
`
`It is readily adaptable to other methods of DNA labelling.
`
`It works as well with short restriction fragments as with longer ones
`and is readily adaptable for the use of oligonucleotides. This greatly
`
`enlarges the number of potential applications.
`
`5
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`10
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`2-.
`
`3.
`
`4.
`
`5. Whereas membranes have a (low) finite capacity for binding DNA,
`
`larger quantities of DNA can be readily handled in the present
`procedure by simply increasing the quantity of immobilised and labelled
`
`15
`
`DNA's. This offers the possibility of either increasing the sensitivity of
`
`the assay or of using a labelled DNA of lower specific activity or of
`
`reducing the hybridisation time still further.
`
`These advantages will now permit the rapid adoption of recombinant
`
`DNA technology to routine laboratories for the identification and character-
`
`20
`
`isation of bacteria, viruses, and genetic disorders in humans, animals and
`plants.
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`wo 86/03782
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`10
`
`we
`
`1.
`
`In a method for the detection of a given nucleotide sequence in a target
`
`polynucleotide, which comprises contacting the target polynucleotide under
`
`hybridisation conditions with (a) an immobilised polynucleotide comprising a
`
`single-stranded nucleotide sequence complementary to, and hybridisable
`
`under said conditions, with a first section of said sequence to be detected,
`
`and (b) a labelled polynucleotide probe comprising a single-stranded nucleo-
`
`tide sequence complementary to, and hybridisable under said conditions, with
`
`10
`
`a second and different section of the sequence to be detected, labelled with a
`
`marker, thereby to form an immobilised hybridisation duplex comprising the
`
`immobilised polynucleotide, the target polynucleotide, and the labelled poly-
`nucleotide, separating the immobilised duplex and determining the presence
`
`15
`
`of
`the marker
`thereon,
`the improvement which comprises using as said
`immobilised polynucleotide, a polynucleotide containing said single-stranded
`
`complementary nucleotide sequence covalently bonded to Solid particles or
`
`beads of a cross-linked macroporous resin.
`
`20
`
`25
`
`30
`
`35
`
`2.
`
`In a method for the detection of a given nucleotide sequence in a target
`
`polynucleotide, which comprises contacting the target polynucleotide under
`
`hybridisation conditions with (a) an immobilised polynucleotide comprising a
`
`single-stranded nucleotide sequence complementary to, and hybridisable
`
`under said conditions, with a first section of said sequence to be detected,
`
`and (b) a labelled polynucleotide probe comprising a single-stranded nucleo-
`
`tide sequence complementary to, and hybridisable under said conditions, with
`
`a second and different section of the sequence to be detected, labelled with a
`
`marker, thereby to form an immobilised hybridisation duplex comprising the
`immobilised polynucleotide, the target polynucleotide and the labelled poly-
`
`nucleotide, separating the immobilised duplex and determining the presence
`
`ll
`
`of
`
`the marker
`
`thereon,
`
`the improvement which comprises using as said
`
`immobilised polynucleotide, a polynucleotide containing said single-stranded
`
`complementary nucleotide sequence covalently bonded to solid particles or
`
`beads of a cross-linked macroporous cellulosic material, said particles or
`
`beads having a particle or head size in the range 5 to 50 p.
`
`3.
`
`A method according to claim 2, wherein the immobilised polynucleotide
`
`
`
`wo 86/03782
`
`PCT/GB85/0059l
`
`11
`
`is covalently bonded to the resin particles or beads by a diazotisation
`reaction between reactive groups on the resin and free amino groups on the
`polynucleotide.
`
`A method according to claim 2, wherein the immobilised polynucleotide
`4.
`is covalently bonded to the resin particles or beads by carbodiimide coupling
`between the phosphate groups at the 5' end of the polynucleotide and reactive
`groups on the resin.
`
`A method according to claim 2, wherein the polynucleotide probe
`5.
`comprises a radioactive marker or label.
`
`A method according to claim 2, wherein the polynucleotide probe
`6.
`comprises an enzyme marker or label.
`
`A method according to claim 2, wherein the immobilised polynucleotide '
`7.
`\
`and the polynucleotide probe are restriction endonuclease polynucleotide
`fragments.
`
`A method for the detection of the abnormal human (sickle cell) B-globin
`B.
`gene, which comprises digesting the gene with a Dde i restriction endo-
`
`nuclease to produce a restriction digest containing as a component a 381 bp
`restriction fragment containing the abnormal sixth codon which is a charac-
`
`teristic of the abnormal gene, contacting the digest containing that fragment
`under hybridisation conditions with (a) an immobilised single-stranded poly-
`nucleotide covalently bonded to solid resin particles or beads, said single—
`stranded polynucleotide being hybridisabie with a
`first section of
`said
`fragment, and (b) with a labelled single-stranded polynucleotide probe hybrid-
`isable with a second section of said fragment, wherein one of said single-
`stranded polynucleotide probe and said labelled single-stranded polynucleo-
`tide is or comprises a 180 bp polynucleotide sequence obtained or obtainable
`by Dde l digestion of normal human B-globin gene and complementary to a
`first section of said 381 bp fragment, and the other is or comprises a 201 bp
`fragment obtained or obtainable by Dde I digestion of normal B—globin gene
`and complementary to a second, different, section of said 381 bp fragment,
`separating the resulting immobilised hybridisation duplex comprising the
`
`10
`
`15
`
`20
`
`25
`
`30
`
`‘35
`
`
`
`WO 86/03782
`
`PCT/GB85/00591
`
`12
`
`immobilised single-stranded polynucleotide,
`
`the said restriction fragment,
`
`and said labelled polynucleotide probe, and determining the presence of the
`
`marker on the immobilised hybridisation duplex.
`
`9.
`
`A method according to claim 8, wherein the said 180 bp polynucleotide
`
`sequence is part of a 341 bp fragment obtained or obtainable by Hinf I
`
`digestion of normal human B-globin gene.
`
`'
`
`10.
`
`A method according to claim 8, wherein the polynucleotide consisting
`
`10
`
`of or containing said 180 bp sequence, as
`
`the case may be, and the
`
`polynucleotide comprising said 201 bp fragment are obtained by the endo-
`
`nuclease restriction of plasmid BFS.
`
`11. A method according to claim 10, wherein the immobilised single-
`stranded polynucleotide is covalently coupled to beads or particles of a cross-
`
`15
`
`linked macroporous cellulosic resin.
`
`12. A diagnostic kit for the detection of a given nucleotide sequence in a
`
`target polynucleotide, comprising as a first component an immobilised poly-
`
`20
`
`nucleotide reagent comprising solid particles or beads of a cross-linked
`macroporous resin having a single-stranded polynucleotide sequencecomple-
`
`mentary to a first section of
`
`the nucleotide sequence to be detected
`
`covalently bonded to said particles or beads, and as a second component, a
`
`polynucleotide probe comprising a single-stranded nucleotide sequence com-
`
`25
`
`plementary to a second, different section of the nucleotide sequence to be
`
`detected, and labelled with a marker.
`
`13.
`
`A diagnostic kit for the detection of the abnormal human (sickle cell)
`
`3O
`
`B-globin gene, comprising as a first component an immobilised polynucleotide
`reagent comprising solid resin particles or beads having covalently bonded
`thereto a first single-stranded polynucleotide sequence, and as a second
`
`component a polynucleotide probe comprising a second single-stranded poly-
`
`nucleotide sequence labelled with a marker, wherein one of said sequences is
`or comprises said 180 bp polynucleotide sequence obtained or obtainable by
`
`35
`
`Dde I digestion of normal human B-globin gene, and the other is or comprises
`
`said 201 bp fragment obtained or obtainable by Dde l digestion of normal
`
`B-globin gene.
`
`
`
`wo 86/03782
`
`PCT/GBSS/0059l
`
`13
`
`14.
`
`A kit according to claim 13, wherein the said 180 bp polynucleotide
`
`sequence is part of a 341 bp fragment obtained or obtainable by Hinf I
`
`digestion of normal human B-globin gene.
`
`15. A kit according to claim 13, wherein the polynucleotide consisting of or
`
`containing said 180 bp sequence, as the case may be, and the polynucleotide
`
`comprising said 201 bp fragment are obtained by the endonuclease restriction
`
`of plasmid BFS.
`
`10
`
`15
`
`20
`
`16.
`
`A kit according to claim 13, wherein said first single-stranded poly-
`
`nucleotide sequence is covalently coupled to particles or beads of a cross-
`
`linked macroporous resin.
`
`17.
`
`A kit according to claim 12, wherein the polynucleotide probe is
`
`labelled with an enzyme, and the kit also contains, as a third component,
`
`means for detecting the enzyme-labelled probe, said means comprising a
`substrate for said enzyme.
`
`18.
`
`A kit according to claim 13, wherein the polynucleotide probe is
`
`labelled with an enzyme, and the kit also contains, as a third component,
`
`means for detecting the enzyme-labelled probe, said means comprising a
`substrate for said enzyme.
`
`
`
`wo 86/03782
`
`PCT/GBSS/00591
`
`EEsme:
`
`32:1EEmacs:
`
`4i;3.3Illlllllllll|llllw
`
`TE23m
`Ill-Ill
`
`2EmEmE:
`
`15
`
`///
`
`
`
`2mmassasm‘.2552
`
`
`
`383.2.:BEBE:
`
`
`
`«EnEEPQ
`
`rilllllllL:a:
`
`:5so2Ea:Nmmgmas95EEa:
`
`EtaCE:
`
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`
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`
`.II]
`
`~E:
`
`SUBSTITUTE SHEET
`
`
`
`INTERNATIONAL SEARCH REPORT
`international Application No PCT/GB 85/00591
`
`I. CLASSIFICATION OF SUBJECT MATTER (it several classification symbols apply, indicate all) '
`According to international Patent Classification (lPC) or to both National Claae7‘cation and IPC
`P 4
`C 12 Q 1/68 // G 01 N 33/546
`G 01 N 33/548
`IC:
`M—
`II. FIELDS SEARCHED
`“M
`Minimum Documentation Searched 7W—
`Classification System I
`Classification Symbols
`
`4
`
`C 12 Q
`G 01 N
`IPC
`
`Documentation Searched other than Minimum Documentation
`to the Extent that such Documents are included In the Field: Searched ‘
`
`
`
`Ill. DOCUMENTS CONSIDERED TO BE RELEVANT.
`
`Category ' I
`Citation oi Document, " with Indication. where appropriate. of the relevant passage: '3
`I Relevant to Claim No. ‘3
`
`I
`
`I
`
`1,2,12
`
`1,2,5,8-
`16
`1,2,5,8-
`16
`
`EP, A, 0070687 (STANDARD OIL.COMPANY) 26
`January 1983, see page 4,
`lines 4—24;
`page 8,
`line 29 - page 9,
`line 32; claim
`1,2
`(cited in the application)
`
`'
`
`I I
`
`X
`
`I
`,
`I
`!
`INC.)
`P,Y§EP, A, 0130523 (MOLECULAR DIAGNOSTICS,
`I
`9 January 1985, see pages 1,2; page 8,
`l
`line 31 — page 9,
`line 2; example
`I
`—_
`P,Y'EP, A, 0130515 (MOLECULAR DIAGNOSTICS,
`INC.)
`I
`9 January 1985, see the whole document
`I
`
`A
`
`A
`
`IUS, A, 4395486 (L.B. WILSON et al.) 26 July
`!
`1983, see the whole document
`l
`--
`i
`gProceedings of the National Academy of Scienc
`l
`USA, volume 80, January 1983
`5
`B.J. Conne
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