CA1266434A

CA1266434A - Use of nonhybridizable nucleic acids for the detection of nucleic acid hybridization

Info

Publication number: CA1266434A
Application number: CA000469904A
Authority: CA
Inventors: Nanibhushan Dattagupta; Peter M.M. Rae; William J. Knowles; Donald M. Crothers
Original assignee: Molecular Diagnostics Inc
Current assignee: Molecular Diagnostics Inc
Priority date: 1983-12-12
Filing date: 1984-12-12
Publication date: 1990-03-06
Anticipated expiration: 2007-03-06
Also published as: ES8609472A1; US4724202A; ZA849622B; JPS60144662A; ES538291A0

Abstract

ABSTRACT OF THE DISCLOSURE

A detection probe comprising a hybridizable single stranded portion of nucleic acid connected with a non-hybridizable, single or double stranded nucleic acid portion, the non-hyhridizable portion preferably including a recognition site for a particular protein.

Description

1 ~66i434 I
1 ~ IE UST;~ IONIIYBRII)I%AE3L,I~ ~[~CI.EIC ACIr)S

2 ¦ 1~'01~ TllE DE'l'}~.CTION OF N~lCI.E':tC l\CID IIYBRIL~IZATION
j ~

3 ¦ This appllcatioll re:Lates to a labeled probe

4 ¦ sui-table for analytical and ~:iagnostic purposes with regarcl ~ ¦ to genetic constitution.
6 ¦ The evaluation of nucleic acid hybridizations is 7 ¦ usually accomplished by detecting radioactivity lntroduced 8 ¦ into a DNA:DNA or DNA:~NA hybrid via one member of a pair of 9 ¦ complementary polynucleotides (-the labelled memher being 10 ¦ designated the probe). ~adiolabelling of the probe is 11 ¦ effected by in vivo or ;n vitro polymerlzation of RNA or DNA
12 ¦ under conditions in which precursors are iso-topically tagged 13 ¦ with 3H, l~C, 125I or 32p, although it is also possible to 14 ¦ label polynucleotides postsynthetically using 125I or lS ¦ 32P-ATP. Each kind of radiolabelling has limi-tations, such 16 as sensitivity of detection, isotope half-life, and hazard-, and it is highly desirable that probe labelling be 18 accomplished without resort to radioactivity.
Available alternatives are (i) at-tachment of haptens such as biotin to nucleic acid precursors, in which 21 case an investiga-tor is required to carry out in vitro 232 ¦ polynucleotide syntheses in order to label a probe, then to 24 ¦ detect the presence oE biotinylated probe in a hybrid ¦ through -the application oE two or more steps; and lii) the 26 ¦ attachment of enzymes to oligonucleotide or polynucleotide 27 ¦ probes, in which case nybrids are detected by their ability I to convert a substrate to an optically or chemically 29 ¦ distinguishable product. Both of these alternatives to 30 ¦ radioactivity invo~ve modera-te -to substantial changes in the chemical ~tructure of probes, 80 that qualitative and/or quantitative effects on hybridization are a posslbillty, if not a reality.
Canadian appllcation Serial No . b54, 9b2, f iled July 25, 1984, discloses a dual hybrldlzation assay conducted with a known and an unknown nucleic acid sample and a nuclelc acld-~ontslning detection probe. Advantsgeously, the known sample, the separation prob0, is immobilized on a solid support and contacted with the unknown and the instant labelled detection probe. The contact is performed under conditions favourable to hybridization. A portion of the unknown nucleic acid hybridizes with the immobilized probe. If the unknown also contalns a nucleotide sequence which is complementary to the nucleotide sequence of the detection probe, a slecond or dual hybridization will then take place by which the detection probe becomes affixed to the solid support. If the unknown nucleic acld lacks the partlcular complementary nucleotide sequence, the detection probe will not hybridize therewith.
Accordin~ly, the extent of the second hybridization, as indicated by the extent of labelling, is an indicator of the presence of the particular nucleotide sequence of interest in the unknown.
It then becomes necessary to determine how much of the second hybridization has ta~en place, i.e., how much of the detection probe is on the immobilized support.
The detection-probe can be labelled with vsrious labels which can be detected in systems that measure specific binding actlvity, fluorescence or enzyme activity.

~ ~266~3~
1 1 Such labels inclucle radioisotopes, fluorescent radicals, 2 ¦ enzymes and haptens. If -too many labels are provicled as in 3 ¦ the case of fluorophores, they nlay interfere with -the second 4 ¦ hybridization. On the other hand, if there are too few

5 ¦ labels, assay is less sensitive.

6 ¦ Tt is, according]y, an object. of the present

7 1 invention to provide a de-tection probe (or the prohe

8 ¦ carrying -the ]abels) which can be usecl in an assay without

9 the disadvantages of radioactivity and withou-t chemical modifieation of the probe components which could interfere 11 with hybridization.
12 It is another object of -the invention to provide a 13 means of labelllng a probe with a large number of readable 14 labels resulting in relatively high sensitivity, without interfering with hybridization.
16 These and other objects and advantages are 18 realized in aceordance with the presen-t invention pursuant 19 to which there is provided a detection probe comprising a hybridizable single strand-ed portion oE nucleic acid which 21 ean hybridize with the unknown, eonneeted with a non-22 ¦ hybridizable single or double stranded nueleie aeid portion, 23 I the non-hybridizable portion advantageously including a 24 ¦ recognition or bincling site for a particular protein. Tf 25 I the non- hybridizable portion is clouble stranded, one of the 26 ¦ strands may be eontinuous, i.e., eovalently assoeiated, with 27 ¦ the hybridizable por-tion.
28 ¦ The hybridizable portion of nueleic acid can be any of -those described in greater detail wi-th regard to the '2~9~i ~
30~D ~ deteet:ion probe of ~pplica-tion Serial No. ~ G3, ~ , as ~2~i643~

1 I ~or exampLe, a nucleotide sequence which is complementary to 2 ¦ the qenomic se-3uence responsible ~or sickle cell anemia.
3 I rhe nuclelc acid o:F the non-hybrldizable portion 4 ¦ can be a natural DN~ sequence or synthe-tic oligonucleo-tide 5 I which contains a highly specific bindiny si.te or sites for a 6 ¦ protein or proteins. The non-hybridizable portion can be 7 ¦ specifi.c for lactose (hereinaf-ter referred to as lac) 8 ¦ repressor protein which binds to an operator J.ocus in the 9 ¦ non-hybridizable portion, which operator must be double

10 ¦ stranded, preferably after hybridization, as hybridization

11 ¦ might sever the bond between opera-tor and repressorO

12 1 Accor.dingly, if the now-immobilized detection probe is

13 ¦ contacted with a solution containing lac repressor protein, that protein will be selectively removed from the solution and will bind to the lac operator. Even i-E the 16 concentration of nonspecific DN~ in the hybridized sample is in 1000-fold excess, the binding to nonspecific sequences is 19 neg]igible. In living cells, repressor proteins bind to their corresponding operator sequences to modulate 21 transcription of a ~ene. When an operator sequence is I covalently attached t.o other sequences, binding of repressor 232 ¦ proteins is still specific for the operator.

24 I In accordance with other aspects of the invention, I there are provided several as~say procedures utilizing -the 26 ¦ novel detec-tion probes.
27 ¦ In one $uch assay for the presence of a particular 28 ¦ nucleic acid nucleotide sequence in a samp].e, either the 29 I sample or a separation probe is immobiliæed on a support and, with a detec-tion probe AS described hereinabove, is .' ~1 _ 4 _ i43~

1 subjected to hybridization, ther~hy aEfi~iny the 2 non-hybridizable portion -to -the suppor-t. ~ protein is bound 3 to the protein recognition site and thereby to the suppor-t.
4 The protein is ]abeled at any s-tage, either before or after binding, and finally -the ]abel is assaye~.
6 In ano-ther assay, af-ter binding the pro-tein and 7 separating the support from the balance oE the material, the 8 support is treated in orcler to dissociate the pro-tein from 9 the hybridized cletection probe and the dissociated protein is then assayed as by reading a label thereon, -the label 11 having been applied at any prior stage.
12 The invention is further described with reference 13 to the accompanying drawing which is a schematic flow sheet

14 of a hybridization-and assay in accordance with the present invention.
16 Referring now more particularly to the drawing, 18 the unknown DNA (to be tested) is processed as by digestion 19 with a restriction enzyme, electrophoretic separation, southern transfer and/or simple denaturation. If not 21 already immobilized, the DN~ is then adsorbed on to a solid 22 ¦ support (e.g., ni-trocellulose paper) directly or by 23 ¦ hybri~iza-tion to a separation probe. The immobilized DNA is 24 I hybridized with a known probe. The known probe (P) has two 25 ¦ regions. The region ps is single stranded and complementary 26 ¦ to a specific gene to be detected and the region pd is a 27 ¦ piece of double or single stranded, non-homologous DNA which 28 ¦ carries the labels by which the labelling reaction will be 29 ¦ detected. pd can be a specific sequence of double stranded DNA which binds a spec;fic protein. For example, the double 1~i6'1L;14 1 stranded I~M~ can be the ]ac proMoter/operator seq~ence and 2 then the protein is lac r~pressor. pd can also be a bind;ng 3 site for a specific antibody. pd can also be a speciEic 4 sinyle stranded, immunogenic polynucleo-tide sequence or poly [d(G-C)] which, when treated with high sa]t, changes its 6 structure and becomes immunogenic in the Z form. I'he pd 7 portion can also be modifiecl with psoralen derivatives or 8 platinum-containing D~JA binding ligands to produce 9 immunogenic sites.
If pd is the lac promoter/operator sequence, pd 11 ~ill bind lac repressor protein after the hybridization.
12 The protein can then be assayed by an antibody or by direct 13 labelling. The double stranded pd portion can also be 14 modified with hapten, e.g., biotin. Then -the hiotinylated hybrid can be detected in a known manner. I'he pd portion can also be rnodified with a number of fluorophores and can 17 be ~ssayed directly.

19 In a specific embodiment involving a lac operator-repressor system, the foregoing process involves at 21 least the following four steps:
22 Step I: Grow bac-teria and isolate lac repressor 23 protein~
24 Step II: Covalently couple the detection probe to lac operator DN~ and clone the adduct -to have a large 26 quarltity of sample;
27 Step III: ~repare lac repressor - FIrl'C or 28 lac repressor-~-galactosidase adduct or anti-lac repressor 29 antibody;
Step IV: llybridiæation and detection of lac operator via label on -the lac repressor.

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1 ThereaEter, the arllount of bound lac repressor 2 protein can be assayed in various ways. For example, 3 antibodies -thereto can be contacted with the bound 4 lac repressor and protein A conjugated wi-th an enzyme can be bound to the antibocli.es. The amount of bound enzyme 6 can then be determined by -the enzyme's catalytlc react.i.on of 7 its substrate in a conventional manner.
8 The amount oE enzyme indicates the amount of lac 9 repressor which, in -turn, indicates the amoun-t of hybridization which occurred ~arlier. Alternatlvely, the 11 lac repressor protein can be fluorescently labelled or 1 labeIled with an enzyme and reacl in a conventional manner.
13 The double stranded region of the de-tection probe 14 can also be specific for galactose repressor protein, lambda repressor pro-tein, catabolite gene activator protein (CAP), 16 Cro protein and the like. ~he foregoing descrip-tion for 18 assay of bound lac repressor protein applies equal.ly to 19 assay for the presence of these proteins. Such proteins can be purified from strains of Escherichia coli. The DM~
sequences to which -these proteins bind have been identified 22 and isolated using recombinant DNA technology. The segment l of E. coli DNA that contai.lls the ~ac repressor binding site 24 ¦ (the lac promo-ter-opera-tor region) is transferred to 25 ¦ recombinant plasmids that include segments o:f human DNA, 26 ¦ such as portions of the gene encoding hemoglobin. These can 27 ¦ be used without Eurther genetic engineering to test :Eor a 28 ¦ numher of hemoglobinopathies, such as some thalassemias and 29 sickle-cell hemoglohinemia. Alternatively, in the dual hybridiæation scheme, two plasmids are used to determine if . - 7 -66~3~

1 ¦ a sample o:E ~NA from a human subject contai.rls the cJenetic 2 ¦ conclition respollsible Eor si.ekle cell hemoglobinemia. One 3 ¦ plasmid is clesignated the separat.ion pro~e. It contains DMA
4 ¦ tha-t is one flank of ~he dimorphic restriction en~yme 5 ¦ cleavage site; i.t is 1.mmobilized as sincJle stranded 6 molecules on a solid support, ancl it is unlabe].led. The 7 seconcl plasmid is designatecl the detee-tion probe. It 8 contains DNA tha-t is -the other flarlk of the dimorphie 9 restriction site, and it has also been engineered to contain a segment of E. coli DN~ that eon-tains the lac promoter/
11 operator region. Through the use of appropriate enzymes, 12 the deteetor plasmid is made par-tia].ly single stranded to 13 the extent that~-globin gene sequences are available for 14 hybridization while lae repressor recognition sites remai.n double stranded and available for protein binding.
16 Read out involving lae repressor pro-tein provides 17 highly speeific recognition of the presence of the detection 18 ¦ probe. It also opens a new set of possibilities for 20 1 solution phase read out because it is possible to release l the repressor-antibody comple~es from the operator DN~ by 21 ¦ addition of a ~-galac-toside sueh as isopropylthio-23 1 galactoside. This allows automated batch or flow system 24 ¦ proeessing.

25 l In the foregoing deseription, -the double stranded 26 ¦ nueleie acid sequence contalned a protein recognition site .

27. ¦ from the outset. Tlowever if it did not contain such a slte 28 ¦ initially, it is possible to modify the DN~ -to ereate 29 l protein or antibody reeognition sites for ease of reaetion 30 ¦ and deteetion.

43~
Such modification can be effacted by contaet with reagents, such as furocoumarins, e.g., angelicins, psoralens, etc. as deseribed more fully in Canadian ayplieation ~erial number 455,96B
filed June 6, 1984. Platinum-containing ligands can be similarly employed. The reagents render the non-hybridizable nucleic acid portion recogni2able by protein. If the non-hybridizable portion is rendered immunogenic, such protein ean be an antibody, i.e., an immunoglobulin, for example, a monoclonal antibody. The antibody can be bound to the non-hybridizable portion in an amount lo corresponding to the amount of furocoumarin creatiny the protein recognition sites. Antibody recognition ~ites ean also be created when pd contains poly~dtG-C)] sequences, and the probe is exposed to high salt concentration.
Alternatively, the protein reeognition ~ite can be on a segment of the non-hybridizable portion other than nucleie acid per se. For example, the nucleie aeid of the non-hybridizable portion can be linked by a member such as a furoeoumarin to a chemical group sueh as ~iotin, the biotin eonstituting the protein recognition site.
The biotin can be assayed in a eonventional manne~, for example, with avidin or an anti-hapten antibody. The furoeoumarin may be linked to a fluorophore, the fluorophore thereafter being assayed for fluorescence.
Labelling with the aforementioned proteins ean be performed either before or after modification of the double stranded nucleie acid, ereferably after.

g _ ll ~.Z~6~39~

1 Sal.ts may also he use(l as a means of modifying the 2 non-hybridi,able port:ion to rende:r ;t protein recogni.zable 3 (e.g., poly [d(G-C)l or po:Ly [cl(CI-m~Cl changf?s to Z-form).
4 Suitab].e salts include sodium ch].oride, other alkali and alkaline earth me-tal soluble sal-ts of mi.neral. acids, 6 spermine or spermiclines, advantageously in concentrations of 7 a-t least about 1~. by welght. ~dvantageously, th.e solvent is 8 water. Both -the salt-modified nucleic acid and the 9 furocoumarin modified nuclei.c acid will be an-tigenic, e.g., will be capable of binding a specific an-tibody whicll can be 11 assayed in conventional manner. ~`or example, as 12 hereinabove, the protein ~ can be conjugated with an enzyme 13 . which functions as the label in suhsequent assay.
14 The invention also extends to assays involving detection probes wherein the non-hybridizable portion has 16 been modified to attach a protein recogni.-tion site, as with 17 a furocoumarin, as a link between the non-hybridizable 19 double or single strand component and the protein recognition. Si te, which can be a hapten or ligand. An 21 - immobilized separat;on probe or tes-t sample is subjec-ted to 22 hybridizing conditions i.n the presence of a detection probe 23 so modified to bind a protein and carrying a label, and the 24 label is.as~ayed. Alternatively, the pro-tein may constitute an antibody and the an-tibody assayed immunologically in 26 conventional manner, wi.lhout eormal labelling of the 27 protein. As another alterrlative, if a hap-ten or ligand 28 is at the protein recognition site, its presence can be 29 assayed. The furocoumar;.n can also link a fluorophore and the fluorophore utiliz.ed as the assayable element.
.

- 10 -- .

~ ~Z~i6~39~

1 The detection probes made and used as describecl 2 above exhibit ~reater sensitivity -than heretofore by vir-tue 3 of -the Ear greater number o~ labels per sinyle stranded 4 nucleic acid prohe molecu]e -than is possible with directly labeling the probe molecules.
6 The lnvention will be further described with 7 reference to the accompanying examples wherein all parts are 8 by weight unless otherwlse expressed.

EX~MPLE 1 12 Step I - Isolatin~ la~ repressor ~ in.
13 _ _ _ __

15 E. co~i strain BMII 461-

16 ~ (lac pro) (~ C1857t68d lac iqz y )/(F lac iq~ y pro ), developed by Muller-~3ill et al. carries a thermally 18 inducible lambda ]ysogen with a lac repressor gene and it overproduces the protein 1000-fold compared to the wild type 21 strain. (Other E. coli strains can also be used to isolate 22 ¦ the pro-tein). The strain is grown substanlially as 23 ¦ described by Muller-llill e-t al. and Platt et al.
24 ¦ [Muller-Tlill et al., Proc._Natl _Acad. Sci.~59 1259 (1968~;
25 ¦ Plat-t et al. in ~. in Mol Genetics (ed.)J. ~liller) CSII, 26 pp. 363-393 (1972)], as follows:
The cells are growll in ~ medium containing 3 28 Bac-totryptone, 2~ Bacto yeast e~tract (both from Difco) 29 and 0.5~ sodiu~ chloride at 32C to an OD 550 of 3. Then the temperature is raised to 44C for 20 minutes to effect thermal induction. The ce]ls are then incubated at 37C

p ~ m ~ ~

~LZ664;i4 l For 5 hollrs~ The cel].s are collectecl from the cul.ture by 2 centrifuga-tion at 600n rpm and storecl fro~.en at -80C.
3,~ 1 lO0 gms of cells are thawed an(l h1.ended in a 4~ ' Waring blender and the supernatant after centrifugation is made up to 100 m]. with a bu:E:Eer comprising 0.2 M Tris HC1, 6 PH 6.9, 0.2 M KCl, 10 mM my acetate, 0.1 m~l di-thiothreitol , 7 5~ (V/v) glycerol) and precipitated hy adding 0.23 g/ml 8 ammonium sulfate. 'rlle precipltate ;.s collected by 9 centrifugation at 10,000 rpm and redissolved in 5 ml of -the foregoing buffer and desalted by exhaus-tive dialysis against 11 a buffer solution comprising 0.12 M potassium phoslhate 12 (P~l7.4~ 0.1 m.~ dithiothreito~., 5~ (V/v! glycerol; 2% (V/v) 13 dimethyl sulfate.
14 The lac repressor protein eluate is final].y purified on a phosphocellulose column using phosphate buffer 16 as above, with a linear yradient of 0.12 to 0.24 potassium 18 phosphate.
19 The purity oE the lac repressor--containing fractlon is checked by SDS-polyacrylamide gel electrophoresis. The act.i.vity of the lac repressor protein 22 is-measured by its ability to bind operator-containing DNA
23 in a known manner. ~he protein can be s-tored a-t -80C until 24 use.

Step II - Cova.1ently coupliny detection probe to 26l~ac_~e~rator DNA _ _ 27 . .
Prepara-ti.on of a plasmid having both multiple 28 .
29copies of the lac repressor protein binding site (lac operator) and a portion o.[ the~ -hemoglobin gene, pursuant ~ '~f~D~r7~a~

. - 12 -~ 3~

1 ¦ to Molecular Cloninc1, Maniat:is et al., Cold Spring Harbor 2 ¦ Laboratory, 1982.

4 ¦ 1. pllW104 is a cleriva-tive of pB~322 tha-t has 4-5 5 ¦ copies of the 203 bp Elae III segment of -the lac operon that 6 ¦ contains the lac repressor binding site. The segment is l tailed with Eco ~I linkers, and tanclem copies are inserted 8 ~ in-to -the Ap Tc vector p~-lWl (a deriva-tive of pBR322 prepared 9 by Elae II digestion to lack the sequence 23h to 2352) at -the Eco RI site.
11 ~ 2. pSS737 is a derivative of pBR322 that has the 12 737 bp ~lu I segment of the human ~ -globin gene that 13 contains about 0.5 kb of the gene and about 0.25 kb of 14 upstream flanking sequence. The segment is tailed Wit}l Eco RI linkers and inserted into the Eco RI site of pB~322.
16 The procedure for putting the lac repressor

17 binding sites and the segment of the ~-globin gene in the

18 single plasmid as in 1 and 2 above, is as follows:

a. Linearize pHW104 with Hind III; treat with 21 alkaline phosphata~e to prevent recircularlzation in step c.
22 ¦ b. Digest pSS737 with Hind III plus Fnu DII;
23 I co]lect the greater than 0.76 kb segment from a preparative 24 ¦ agarose gel.
25 ¦ c. Liga-te the products of steps a and b, then l fill in free Elind III ends using the Klenow fragment of 27 ¦ DNA polymerase and de(!~yribnuc]eotide tripho.sphates.

28 l d. Blun-t-end ligate (c) molecules to make 29 circular plasmicls, then transform E. coli cells to ampicillin res;stance.

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e. Collect a number of ApR colonies and grow cells for the minilysate production of small amounts of plasmid.
f. Check the pla~mids for compos~tion by restriction enzyme digestion.
The desired plasmid has:
i. a singla Hind III slte;
ii. Eco RI segments of 2.2, 0.74 and 0.21 kb;
111. dlgQstlbility by H~t II;
~ v. Desirably a Cla I segment of about 0.75 kb, depending on the orlentation of the globin gen lnsert.
The separatlvn and detection probe for dual hybrldization anaylsis of sicXls cell defect are dlsclosed in detall in said Application Serial No.
454,942.
3. Vse of a plasmid having both multiple copies of the lac repressor bindlng sits and a portion of the ~ - hemoglobin g9n8 as a hybridization probe:
For the plasmid to be a useful probs for the detection of~- globin gene sequsnces ~n a sample of DNA, the globin gene portlon of the plasmid must be single stranded so that ln a subsequent test it can hybridize to a sample of denatured DNA, and the lac operator region must be double stranded to allow blndlng of the lac repressor protein.
To achisve this, the plasmid product of (2) ls llnearized using Hlnd III, then ls sub~ected to a controlled dl~astion by exonuclease III (~ exonuclease or T4D~A polymerase can be s~milarly employed~. Such treatmsnt makss most or all of the globin gene portion single stranded, ~Z~6434 1 leaving most o~ the rest of the plasmid, including the 2 copies of the lac operator region double stranded.
3 Alternatively, pairs of pEMBII plasmids (avail.abl.e 4 from the Europe~n ~olecular Biology Laboratories, IIeidelberg) can be used. These pl.asmids con-tain a portion 6 of the F1 phage genome, so that they behave like phage ~13 7 in producing single stranded DN~ molecules. Unl.ike with 8 M13, however, it i.s possihle with pEMI3L. to collec-t both ~ complemeIltary stands of a plasmid in pure form simply by having the F1 portion of the pEMBr. genome in dif.Eerent 11 orientatlon in two strains; i.t is tlle orientation of -the F
12 genes in the plasmi.d that determines which o-E -the two 13 strands of the plasmid DNA will be secreted :Erom infected 14 bacteria as single stranded DNA phage.
For example, one plasmid, pE~IBL8(-l), i.s engineered lb to contain tandem copies of the lac repressor binding site 18 plus a portion of the ~-hemoglobin gene; another plasmid,

19 pEMBL8~-), contains iust the tandem copies of the lac repressor binding-site. The single stranded DN~ of pE~lBI.8(~) is- hybridized to a sample of unknown DN~, and contact is macle through sequence llomology between the globin 23 gene portion of the probe and complementary sequences in the 24 sample. The lac operator portion of the probe is made double strancled for lac repressc>r hinding by the annealing 26 of pE~IBL8(-~ to the pE~lBI,8(~ r.ample DN~ comple~.
27 It is possi.bl.e to carry out such reactions wi-th 28 the replica-tive (but not the sing].e stranded phage) form of 29 M13 as well as with any pl.asmid DNA, hut one has either to separate the complementary stancls, or take considerable loss ~6~39~

1 in hybricli7.ati.0n ef lciency hy havi.ng hoth strands o:E a 2 plasmicl present in a hybridiæation mixture, where they can 3 undesirably self-anneal S-tep III - (a) r,abel].inc3 of the protei.n 6 _ with Eluorescein __ _ 7 E'luoresceinisothiocyallate (FITC) is dissolved in 8 ethanol (5 mg solid/ml). To 2 ml. of a 5 mg/ml. protein 9 solution from (I), 0.5 ml carbonate buffer (1 M NallC03-Na2C03 buffer pH 9) is added, followed by 50 ~ 'ITC solution. The 11 mixture is shaken well and the free FITC is chrc?matographi-12 cally separa-ted :Erom the bound molecules on a Sephadex G50 13 column using a buffer comprising 10 m~l Tri.s, 1 mM EDTA, 14 50 m~lI<Cl, pH 7.4. The labelled protein is collected in the void volume.

17 (b) l,abelling with ~-galactosidase enzyme .19 Lac repressor protein from I and~,-galactosidase

20 (1:1 molar rati.o~ ~alkaline phosphatase, horseradish peroxi-

21 dase react similarly) are mixecl in pho.sphate buffer and

22 glutaraldehyde is added to final concentra-tion of 0.2%.

23 Reaction i5 al.lowed to proceed for 4 hour~s. The protein .

24 mixture ls dialyzed against the same Tris EDTA buffer as in (a).

27 Step IV - llyhridizatic)rl and detec-tion via 28 labe]s on the_Laç repressor___ 29 Hybridizatlon is done by fixing the separatior 31?~ ~ probe to a so].id support accordillg -to the example of . ~5~,qY~
App]ication Seri.a]. Mo. *~ fi-3~ u~a, using as -the , .

~2~6~34 l ¦ detection probe t~lat probe produced in TI whi.ch ca.rri.es 2 ¦ the nonhomologous DN~ whi.cll is the lac repressor protein 3 ¦ bincding site. After hybrit~i7~ation, the solid support is 4 ¦ washed with BSA solut.ion 1% ~/v in Tris-E~TA bu:ffer as in 5 ¦ Example ]., Step IIT~a~, and then lac repressor, 1.abelled 6 l as in III(a), (b) or (c), is addecl. The bound cepressor is 7 ¦ assayed optically (:in the e~ample of fluor.escein labe].l.ec1 8 ¦ repressor) or en~ymatica]ly (in the exar(lple of enzyme 9 ¦ labelled repressor).
10 l 12 I ~XAMPI~ 2 13 ¦ E~ybridized probe of Example l earrying the 14 ¦ lae repressor protein can be assayed immunochemically 15 ¦ as follows:
16 ¦ a) Purified lac repressor protein from ~xample l 17 is mixed l:l with Freund's complete adjuvant and in,eeted 18 into miee (25~y protein into both hind foot pads) or rabbits (500,~g subcutaneollsly). One mon-th later the polyelonal an-tibody response is titered and the antiserum 21 from animals with strong responses is colleeted and used for 2~ I the immunoassays.
` 24 ¦ b) To the hybricl compl.ex contai.ning the lac l repressor protein oF ~xample l, specifie dilutions of -the 26 ¦ antiserum of (a) are incubated for l hour at room 27 I temperature. ~nbourld antiboc1ies are washed 3 times w.ith a 28 l buf,er solution comprising 5 mM NaH2PO~, l50 mM NaCl (pll 2 ~ ¦ 7.4) and 0.04% Triton X-l00. Protein ~ covalently eoupled ¦- to horseradish peroxidase (Sigma Chemiea] Co. p 8651!

.. ¦ ~ T 2 ~

~266~39~

diluted 1:8000 in PBS as above, i8 incubated with the hybridi7ation complex of Example 1 for 30 mlnutes at room t~mperature and wash2d 3 tlmes with the aforementioned buffer. The substrate o-phenylenediamine ls c~trate buffer containing H202, pH 5.6, is added and the enzymatic reaction product is measured at 492 nm. The amount of bound repressor is determined by comparison to standard quantitation curves.

The double stranded portion of the detection probe of Example 1 can be modified to bind a specific antibody as follows:
The detection probe is dissolved in 10 n~l Tris 1 mM E~TA buffer and mixed with biotin-psoralen adduct as known. The mixture is irradiated with 360 nm light at room temperaturs for 40 minutes. After the reaction, the sample is dialyzed against the hybridizatlon buffer of Example 1, to exclude unreacted biotin-psoralen adduct.
The biotln-containing detection probe is then hybrldized as in Step IV and the hybrid is assayed for the presence of biotin in known manner employing FITC-labelled avldin.
It is understood that the speclfication and examples are illustrative, but not limiting to the present invention and that other embodiments within the spirit and ,, ~ .

;6~3~

1 ¦ scope oF thP lnvelltlon wil 1 suqgest themselves to those 3 sk led in th- ~rt.

1 ~ .

3 . .

8 . . .

O

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE
IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A detection probe comprising a hybridizable single stranded portion of nucleic acid connected to a non-hybridizable, single stranded nucleic acid portion, the non-hybridizable portion including a recognition site for a particular protein, wherein the non-hybridizable portion has been modified to create the protein recognition site, wherein the modification is effected by connecting to said non-hybridizable portion a moiety selected from the group consisting of furocoumarin, phenanthridinium and a platinum-containing ligand.

2. A detection probe according to claim 1, wherein the hybridizable portion is continuous with one of the strands of the non-hybridizable portion.

3. A detection probe according to claim 1, wherein the hybridizable portion is complementary to the genomic sequence responsible for sickle cell anemia.

4. A detection probe according to claim 1, wherein the non-hybridizable portion is double stranded and is specific for lactose repressor protein.

5. A detection probe according to claim 1, wherein the non-hybridizable portion includes a recognition site for galactose repressor.

6. A detection probe according to claim 1, wherein the non-hybridizable portion includes a recognition site for lambda repressor.

7. A detection probe according to claim 1, wherein the non-hybridizable portion includes a recognition site for catabolite gene activator protein.

8. A detection probe according to claim 1, wherein the non-hybridizable portion includes a recognition site for Cro protein.

9. A detection probe according to claim 1, wherein the modifier is a furocoumarin carrying a protein binding site.

10. A detection probe according to claim 1, including a protein bound to the protein-specific site of the non-hybridizable portion.

11. A detection probe according to claim 10, wherein the protein carriers a label.

12. A detection probe according to claim 10, wherein the protein is an antibody specific for a modified double stranded portion.

13. A detection probe according to claim 10, wherein the protein is an immunoglobulin.

14. A detection probe according to claim 13, including a labelled protein bound to the immunoglobulin.

15. A detection probe according to claim 10, wherein the nucleic acid is antigenic and is bound through an immunoglobulin to a labelled protein.

16. A process for conducting an assay for the presence of a particular nucleic acid sequence comprising immobilizing either (a) a separation probe or (b) an unknown sample to be assayed on a solid support, subjecting the support to hybridizing conditions in the presence of a detection probe according to claim 1, binding to the non-hybridizable portion of the probe a protein specific thereto, labelling the protein at any stage in the process, and assaying the label.

17. A process for conducting an assay for the presence of a particular nucleic acid sequence comprising immobilizing either (a) a separation probe or (b) an unknown sample to be assayed on a solid support, subjecting the support to hybridizing conditions in the presence of a detection probe according to claim 1, and assaying the label.

18. A process for conducting an assay for the presence of a particular nucleic acid sequence comprising immobilizing either (a) a separation probe or (b) an unknown sample to be assayed on a solid support, subjecting the support to hybridizing conditions in the presence of a detection probe according to claim 1, reacting the modified non-hydridizable portion with an antibody specific therefor, and assaying the antibody.

19. A detection probe comprising a hybridizable single stranded portion of nucleic acid connected with a non-hybridizable single or double stranded nucleic acid portion, the non-hybridizable portion carrying a fluorophore.

20. A detection probe according to claim 19, wherein the fluorophore is coupled to the nucleic acid of the non-hybridizable portion by a furocoumarin.

21. A process for conducting an assay for the presence of a particular nucleic acid sequence comprising immobilizing either (a) a separation probe or (b) an unknown sample to be assayed on a solid support, subjecting the support to hybrizing conditions in the presence of a detection probe according to claim 9, the furocoumarin carrying a hapten or ligand, and assaying the hapten or ligand.

22. A process for conducting an assay for the presence of a particular nucleic acid sequence comprising immobilizing either (a) a separation probe or (b) an unknown sample to be assayed on a solid support, subjecting the support to hybridizing conditions in the presence of a detection probe according to claim 20, the furocoumarin carrying a fluorophore, and assaying the fluorophore.

23. A process for conducting an assay for the presence of a particular nucleic acid sequence comprising immobilizing either (a) a separation probe or (b) an unknown sample to be assayed on a solid support, subjecting the support to hybridizing conditions in the presence of a detection probe according to claim 19, and assaying the fluorophore.

24. A process for conducting an assay for the presence of a particular nucleic acid sequence comprising immobilizing either (a) a separation probe or (b) an unknown sample to be assayed on a solid support, subjecting the support to hybridizing conditions in the presence of a detection probe according to claim 1, binding to the non-hybridizable portion of the probe a protein specific thereto, labelling the protein at any stage in the process, separating the support from the balance of the material, dissociating the bound protein from the hybridized detection probe, and assaying the dissociated protein.

25. A test kit for assaying for the presence of a particular nucleic acid sequence comprising a detection probe according to claim 1, and a protein recognizable by the protein recognition site of the non-hybridizable portion.

26. A test kit for assaying for the presence of a particular nucleic acid sequence in a sample comprising a detection probe according to claim 1, a separation probe comprising a single stranded nucleic acid immobilized on a support, the single stranded nucleic acid and the hybridizable portion of the detection probe being hybridizable with the sample, and a protein recognizable by the protein recognition site of the non-hybridizable portion of the detection probe.

27. A detection probe according to claim 9, wherein the protein binding site comprises biotin.

28. A detection probe according to claim 1, comprising wherein the non-hybridizable portion is modified by a fluorescent moiety.

29. A detection probe according to claim 28, wherein the fluorescent moiety is linked to a furocoumarin.

30. A detection probe according to claim 28, wherein the fluorescent moiety comprises fluorescein.

31. A method for determining whether the DNA contained in a test sample includes a particular nucleic acid sequence, comprising the steps of:
a) extracting nucleic acids from the test sample, b) digesting the extracted nucleic acids with a restriction enzyme thereby to cleave the DNA or not at a particular sequence, depending on whether or not a restriction enzyme recognition site is present in the sequence, c) treating the product of step b) to form single-stranded nucleic acids, d) contacting the single-stranded nucleic acids produced in step c) with a first and a second polynucleotide probe, each of said probes being complementary to respective first and second portions of said sequence to be detected, said first probe being in solution with said test sample, said first probe being a probe according to claim 1, said second probe being attached to a solid support, the two portions being non-overlapping and immediately adjacent to the restriction site, the restriction site being between the first and second portions, such contact being performed under conditions favourable to hybridization of said first and second probes to said sequence to be detected, hybridization with both of said probes being dependent upon whether in step b) restriction did not occur, said first probe being incorporated with a distinguishable label, e) separating, by means of said second probe, (i) any resulting dual hybridization product comprising said sequence to be detected hybridized to both said labeled first probe and said second probe, from (ii) any unhybridized and singly hybridized labeled first probe, and f) detecting by means of said label any of said separated dual hybridization product which may be present.