CA1258624A - Photoimmune detection of dna and rna - Google Patents

Abstract

PHOTOIMMUNE DETECTION OF DNA AND RNA
Abstract of the Invention A photoimmune method for detecting a particular polynucleotide sequence in a nucleic acid-containing sample is provided. A single-stranded polynucleotide probe substan-tially complementary to the polynucleotide sequence to be detected is irradiated with ultraviolet light to induce formation of UV-photoproducts, principally pyrimidine dimers.
The UV-labelled probe is contacted with denatured nucleic acid from the test sample under hybridizing conditions.
Duplex formation is detected with anti-UV-nucleic acid antibodies which bind the irradiated probe. The antibodies are labelled with a signalling means. A method for detect-ing/quantifying total DNA or RNA in a sample is also provided, as are kits for detecting particular nucleotide sequences, or for detecting total nucleic acid.

Description

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Field Of The Invention The present invention relates to an assay method for detection of speciic nucleotide sequences in DNA or RNA, and to kits useful for performing such assays.
15Background of The Invention Molecular hybridization is extremely useful in the detection of specific nucleotide sequences in genetic materials. Generally, single-stranded chromosomal DNA
from a test specimen is denatured and attached to a DNA-_ 20 binding membrane or filter support. The membrane-filter is brought into contact with a labeled single-stranded polynucleotide probe under conditions promoting hybridization of complementary DNA sequences. ~ouble stranded 'duplex~
or "hybrid~ molecules are detected by a variety of techniques, depending on the nature of the particular label used. In order for hybridization to occur, the probe-molecules must contain sequences substantially complementary to those sequences in the nucleic acid to be detected. An example of such a hybridization system is U.S. Patent No. 4,3581535 to Falkow et al.
Until recently, hybrid molecules have been detected exclusively with radioisotopically labeled probes. The presence of hybrids is detected by scintillation counting or autoradiography, thereby providing a quantitative assay for the presence of the specific DNA of interest. Although autoradiography is a senstitive method, it can be -time consuming. Moreover, hybrid-detecting methods involving .

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the use of radioisotopes must be performed under stringent safety precautions. Radioisotopes are extremely expen-sive, and in some cases have a limited shelf life due ~o rapid disintegration.
6 A currently used method for radioactivly labeling DNA probe molecules incorporates 32p_, 14C_ or 3H-con-taininy nucleotides by the "nick translation" method of Rigby et al., J. Mol. siol. 113:237 (1977). According to this technique, nuclease treatment of the probe nucleic acid is necessary to nick or open gaps in one of the strands. A radiolabelled nucleotide is then inserted into the DNA with a polymerase.
A more recent innovation in molecular genetic probe technology involves utilization of the nick-translation technique to incorporate biotinylated dUTP into probe DNA.
The nick-translation technique requires nuclease treatment of the template nucleic acid to open gaps in one of the strands. A radiolabelled or biotin-substituted nucleotide is inserted with a polymerase. The duplex is _ 20 then split with the thus-labelled strand being used as a probe.
Biotin-labeled DNA may be detected by a soluble complex of biotin-bound horseradish~peroxidase and strepta vidin, or by means of a biotin-specific antibody, followed by a secondary fluorescein-labeled antibody.
A more recent technique dispensing with the need for nick-translation is described in European Patent Application Publication 1280l8. Nucleotides of the probe molecule are modified in situ, for example, by alkylation. Antibodies to the alkylated probe are then used to detect the formation of duplexes.
Yet another chemical-modification approach is disclosed in European Patent Application Publication 122614. A chem-ically-labeled nucleotide, e.g., a biotinylated nucleotide, capable of acting as a substrate for terminal deaxynucleotide transferase, is polymerized onto the terminal end o~ the .
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probe molecule. A polymer with a biotin-containing analog of TTP is ~ormed on the 3'-OH terrninus oE the probe molecule.
Antibodies to the probe molecule may be used to detect the presence oE hybrid molecules.
6 The above methods, relyin~ on the insertion of radio-labeled or chemically-modified nucleotides, on chemical modification of probe DNA molecules, or on termlnal poly-merization oE biotinylated nucleotides, are costly and time consuming. Although non-isotopic labelling of nucleic acids has been successfully employed in molecular hybridi-zation and diagnostic tests, Langer et al., Proc.
Natl. Acad. Sci. USA, 68:6633 (1981), Singer et al., Proc. Natl. Acad. Sci. USA, 79:7331-35 (1982), ~utchinson et al., J. Cell. Biol., 95:609-618 (1982), the key sub-stance, biotin~lated dUTP, is a high-cost product because of its intricate chemical synthesis.
Antibodies to UV-irradiated DNA have been prepared by Mitchell et al., Biochem. siophys. Acta, 655:54-60 (1981) and Eggset et al., Carcinogenesis 4:745-750 (1983).
However~ such antibodies haYe not heretofore been used in conjunction with molecular probes to detect the presence of specific nucleotide sequences.
What is needed is a simple but sensitive method for detecting DNA which dispenses with the need for time con-

2~ suming and costly radiolabelling or intricate chemicalsynthesis of specialized nucleotides.
Summary Of The Invention A method for detecting the presence of specific nucleic acid in a sample is provided. A polynucleotide probe which will hybridize with the suspect nucleic acid is prepared.
The probe is labelled by inducing the formation of UV-photo-products by exposure to ultraviolet radiation. The photo-products are recognizable by antibodies to the UV-labelled probe. The labelled probe is then contacted with substantially sin~le-stranded nucleic acid from the sample under hybridizing conditions. ~ybrid complexes are detected with anti-UV-nucleic acid antibodies labelled with a signalling means. The anti-bodies bind the W -labelled probe -to lndicate the presence of the suspect nucleic acld in the sample.

S In one embodiment of the invention, the labelled probe includes a polypyrimidine nucleotide sequence or "tail" at the

3'-OH terminus. uv-photoproducts, principally pyrimidine dimers, are created in the tail by exposure to UV-radiation. The tail is generated by cloning the probe directly into a polypyrimidine nucleotide sequence or by polymerizing pyridmidne nucleotides onto -the 3'-OH terminal end of the probe with the enzyme terminal transferase.

In another asepct the present invention also relates to a method Eor detecting to-tal nucleic acid. A sample purportedly containing nucleic acid is irradiated with ultraviolet light to form UV photoproducts in nucleic acid contained within the sample. The irradiated sample is contacted with anti-UV-~ucleic acid antibodies. A signalling means associated with the antl-bodies signals their binding to nucleic acid in the sample.

In a still further aspect the invention relates to akit for detecting the presence of specific nucleic acid in a suspect sample. The kit contains a supply of UV-labelled nucleic probe molecules containing UV-photoproducts. The probe is selec-ted so as to hybridize with the nucleic acid to be detected. The kit further contains means for contacting the suspect sample with the UV-labelled probe to from hybrid complexes. The kit contains a supply of anti-UV nucleic acid antibodies which bind the UV-labelled probe. The anti-W -nucleic acid antibodies are labelled with a signalling means. The kit may contain a means for measur-ing the signal to indicate the presence of or extent of the nuc-leic acid being detected.

In yet another aspect, the invention provides a kit which may be used to quantify total DNA in a tes-t sample, inc-X

ludes a supply of anti-UV-DN~ antibody, a panel of DNA-containing ~amples of known DNA concentrations as standards, a signall1ng means for indicating binding of anti-UV-DNA antibody, and means for comparing 2~

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the le~Jel of the signal yenerated by DN~ in the test sample witl~ the levels o~ signa] getlerated by the various members of the standard panel.
It is there~ore an object of the invention to pro~
vide a method for detectin~ or quantifying the pres~nce of specific nucleic acids in a sample.
It is an object of the invention to provide a method for detecting specific nucleic acids without the need for radioactive labelling or chemical synthesis of biotinylated or other synthetic nucleotides.
It is an object oE the invention to provide a method for detecting nucleic acids which may be used to detect the presence oE organisms containing either single-stranded or double-stranded genetic material.
It is an object of the invention to provide a method for quantifying total DNA in a sample.
It is a Eurther object of the in~ention to provide a test kit for the detection or quantification of specific nucleic acids.
It is an object oE the invention to provide a kit for detecting the amount oE total DNA in a test sample.
These and other objects are apparent from the following disclosure.
Brief Description of the Figures 2~ Figur~ l~contain chemical structures of W -induced DNA-photoproducts which may be generated in probe molecules according to the present invention.
Figure 2 is a schematic sequence illustrating the process of the present invention for photoimmune detection of DNA wherein a single-stranded DNA-probe has been extended with a poly-dT tail sequence before UV-irradiation to increase the number of UV-photoproducts for antibody binding.
Figure 3 is a photograph of the hybridization of a representative UV-irradiated DNA probe to homologous DNA
which is attached to a membrane filter. Hybrids were detected with rabbit anti-UV-DN~ antibody and biotinylated ~.

~oat-antl-rabbi-t IgG, followed by incubation with strep-tavidin an~ bio-tinylated alkaline phospha-tase.
Figure 4 is a photograph o~ -the hybridization of a UV-irradiated bac-teriophage Hyl7 DNA-probe to different con-6 centrations o~ single-stranded Hyl7 DNA bound to a fil-ter membrane in the presence of the negative controls bacterio-phage lambda-DNA and calf-thymus-DNA.
Figure 5A is a photograph of a conventional ethidium bromide staining of restriction enzyme-di~ested DNA following agarose gel electrophoresis.
Figure 5B is a photograph of the same DNA fragments following alkaline denaturation, transfer to a filter-membrane by electroblotting, and incubation with anti-UV~
D~A-antibody conjugated to horseradish peroY~idase.
Detailed Description of the Invention By 'IUV-photoproduct'' as used herein is meant a nucleo-tide sequence which has undergone chemical modification in one or more nucleotides by exposure to ultraviolet radiation.
By "UV-nucleic acid" or "UV-DNA" is meant nucleic acid or DNA which has been exposed to ultraviolet radiation thereby causing the formation of UV-photoproducts.
By "anti W -n~cleic acid antibody" or "anti- W -~NA
antibody" is meant antibodies which bind UV-nucleic acid or UV-DNA but not normal nucleic acid or normal DNA.
2~ The present invention is based upon a form oE ln s _ -labelling o~ DNA by means of W -light which occurs in a one-step process, as distinguished from the incorporation of radiolabelled or chemically-labelled nueleotides using the nick-translation method, or the terminal polymerization of such labelled nucleotides to probe molecules.
A polynueleotide probe molecule is prepared whieh eon-tains a nucleotide sequence substantially complementary to the nucleotide sequence of the unknown genetic material to be deteeted. The probe comprises a single strand, or partially 3~ denatured double strand, of nucleie acid. rrhe probe may be DNA or R~A, which is extracted from the relevant genetie material and eloned or synthesized chemically when the nucleo-tide sequence is known. Aecording to ~.S. 4,358,535, probe 1'~5~

molecules may be obtalned from messenger RNA, from cDNA
generated by reverse transcription of messenger RNA, or from cleava~e o~ -the genome, followed by cloning o~ the probe molecule in accordance wi-th exis-ting techniques, e.g. Mani-6 atis, T., et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory (1982), p. 187, 211.
The DNA-probe molecules are irradiated with a relatively low dose of ultraviolet radiation of between 200-400nm wavelength, for example, 2500 J/m2 at 254nm, in order to form UV-photoproducts. Most of the photoproducts thus formed are pyrimidine dimers, but a few saturated pyrimidine products also occur. See Figure 1. Binding occurs between ,~ the C-5 position in a first pyrimidine and the C-5 position in a neighboring pyrimidine base, ~-between the C-6 position in the first pyrirnidine base and the C-6 position in the neighboring pyrimidine base. Binding may also occur between the C-6 position of the pyrimidine base closest to the 3'-OH
terminal end of the probe and the C-4 position of the neighboring pyrirnidine base. Irradiation can also include saturation of tlle double bond between the C-5 and C-6 position of any pyrimidine ring through UV-induced uptake of hydrogen and/or hydroxyl. The DNA probe molecules bearing the aforementioned photoproducts are potent antigens to which antibodies may be raised.
26 Photoproducts, can, as an alternative, be produced by W-light in combination with a photoreactive chemical compound such as furocoumarin.
The sample material to be assayed comprises nucleic acid, either single or double stranded. When assaying or nucleic acid form organisms containing double-stranded nucleic acids, a denaturing step is required. Denaturing techniques are well-known in the art and do not form part oE the present invention. See, for example, Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory -(1982), pO 314, 321, 383; Gergen et al., Nu~leic Acids Res. 7 2115 2136 (1980). Heat denaturation at 95-100C for five minutes, followed by rapid chilling is the preferred method oE denaturation.
) The irradiated probe-D~A is incubated with the sample nucleic acid material containing the purported nucleotide sequence. According to one technique, the sample nucleic acid is a~fixed to a supyort material capable of binding nucleic acid. Cellulosic supports are preferred, partic-ularly nitrocellulosic Eilter papers, which readily bind DNA. One such material is the "Gene Screen" hybridization transfer membrane available from New England Nuclear. It should be understood, however, that attachm~nt to a solid support material is merely one way of visualizing hybridi-zation, it being contemplated that the hybridization process may also be performed in solution.
~ybridization per se is a well-known technique and is described in the literature. See, ~or example, Molecular Cloning, supra; U.S. 4,358,535. The particular hybridiza--tion technique utilized will depend upon the nature of the sample material.
UV-photoproducts in DNA do not decrease hybridization capacity significantly when the probes are greater than 100 bases. Smaller probes wlll also hybridize well with a low UV-dose.
The formation of hybrid complexes between the probe-nucleic acid and nucleic acid in the test specimen is detected by anti-UV-nucleic acid antibodies which are labelled with a suitable signal means. Anti-W -nucleic acid antibodies may be produced according to ~itchell, Do L. and Clarkson, J. M., Blochem. Biophys. Acta, 655~ 54-60 (1981) or Eggset et al, Carcinogenesis 4:745-750 (1983), which disclose immunization of rabbits with single-stranded UV-irradiated calf thymus DNA conjugated to methylated bovine serum albumin. Antibodies with specificity against un-irradiated DNA may be removed by affinity column c~ron~togra~hy through cyanogen bromide activated Sepharose ~ (Pharmacia, Sweden) coupled to un-irradiated DNA (Eggset et al, Supra).
We have found'that the unbound antibody fraction spécifi-cally binds UV-irradiated DNA with ten times greater affinity against single-stranded DNA compared to double-stranded DNA.

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Polyclonal antibodies against UV-nucleic acid pre-pared in this manner, or specific monoclonal antibodies thereto, may be used to detect molecular hy~ridization according to the present invention.
6 The antibody is labelled with a signalling means. The signal is detected or measured as an indication of the presence or extent of the suspect nucleic acid in the sample. Suitable signalling means used to label the anti-UV-nucleic acid antibodies include chemical labels which are detected on the basis of their own physical properties.
Such chemical labels comprise, by way of illustration, and not by way of limitation, color-indicating compounds, particularly fluorescent dyes such as fluoroscein and rhodamine, or reagents of high electron density such as ferritin, hemocyanin and colloidal gold. Alternatively, the signalling means may comprise a substance which is detected by its binding or reactive properties, such as an enzyme which catalyzes a reaction leading to the generation of detectable reaction products. Examples of such enzymes _ 20 comprise peroxidase and al~aline phosphatase.
Most advantageously, a secondary antibody w7nich binds to the anti-UV-nucleic acid antibodies is labelled with the signallin~ means. ~or example, where the anti-UV-antibodies are raised from rabbits, the secondary antibody 2~ may comprise swine-anti-rabbit I~G, conjugated to horse-radish-peroxidase. By the addition of suitable substrates, in this case hydrogen peroxide and diaminobenzidine (DAB), ' the enzyme will give a color reaction.
The secondary antibody may comprise a biotinylated antibody which binds the anti- W-nucleic acid antibody and further binds streptavidin, which may in turn be coupled to a signalling means.
The anti-nucleic acid antibodies may be added to the system following a suitable incubation period to allow the probe molecules to hybridize. Alternativel~ the anti-bodies may be permitted to bind to the probe prîor to hybridization.

In one embodiment of the invention, a polynucleotide probe is ~repared whi~h includes a polypyrimidine nucleo tide sequence at the 3'-Orl terminus oE the probe to increase the sensitivity oE the assay. Roly-dT is the 6 preEerred pyrimidine nucleotide. Polypyrimidine nucleo-tide "tail" sequences may ~e thus generated by cloning the probe directly into a polypyrimidine sequence. The probe is inserted into a c]oning vector adjacent to a poly-dT sequence. Useful cloning vectors for this purpose include single-stranded phage vectors such as ~-13 which are able to s~rete phage particles containing poly dT-tailed DNA probe molecules in single-s-tranded form, ready for use.
The tail sequence may also be generated by enzymati-cally polymerizing pyrirnidine nucleotides onto the 3'-O~
terminal end of the probe with kerminal déoxynucleotide transferase. A typical tail prepared in this manner comprises between about 600 and about 2000 nuclectides.
The creation of polypyrimidine nucleotide tails on the probe molecules results in increased sensitivity in the assay technique. Probes with poly-dT tails constitute a larger target for generation of UV-photoproducts. More-over, after the probe molecules have been hybridized to form duplexes with complementary DNA, the single-stranded 26 poly-dT tails remain unhybridized, and protrude from the duplex molecules. The photoproducts contained in the tail are easily accessible to anti-UV-DNA antibodies, and provide improved antibody binding, making the test system more sensitive.
30The present process of photoimmune detection of hybrid molecules is illustrated diagramatically in Figure 2:
(1) Preparation o probe-DNA: The DNA containing the desired probe sequences are cleaved with restriction enzymes or fragmented mechanically to create more ends. The probe-DNA
is then denatured at 100C for five minutes, followed by cooling on ice (Fig. 2-1)D
(2) (Optional) The probe-DNA is treated with terminal transferase and dTTp to generate a poly-dT-tail sequence at . .

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the 3'-0~l end of the molecule (Fig. 2-2). ~lternatively, the tail is put into position by cloning the probe directly into a poly-dT-sequence.
(3) The probe-DNA is labelled by W -irradiation 6 (~54 nm) which leads to tl-e formation of UV-photoproducts that are reco.~nizable by antlbodies (Fig~ 2-3).

(4) DNA-D~A- (or DNA-RNA-) hybridization: The labelled probe is incubated with a membrane containing samples of single-stranded DNA (or RNA), followed by washing (Fig. 2-4).

(5) The membrane with the hybridized probe-DNA is incubated with anti-UV-DNA-antibody (Fig. 2-5). Excess antibody is washed off.

(6) The co~plex thus formed ls incubated with secondary antibody to the anti-~V-UNA-antibody, e.g. swine-anti-rabbit IgG, linked to norseradish-peroxidase (Fig~
2-6). Excess antibody is wasned off.

(7) The membrane is developed by means of hydroyen peroxide and diaminobenzidine (Fig. 2-7).
~ In addition to identification of specific nucleic acid sequences using 2rohe ~olecules, the present invention may also be used to detect total nucleic acid. A sample purportedly containing nucleic acid is irradiated with ultraviolet light to form UV-photoproducts in the nucleic 26 acid. The sample is then contacted with antibody to W -irradiated nucleic acid. A signalling means associated with the antibody signals the binding oE antibody, thereby indicating the presence of nucleic acid in the sample. The various signalling means employed to detect the presence of antibody binding to probe/nucleic acid hybrids may also be used in the present method for detecting total nucleic acid.
The system offers an extremely sensitive method Eor measuring low nucleic acid concentrations in solution as well as in gels.
According to one embodiment, a sample to be analyzed is irradiated by W-light. The DNA in the sample is denatured and attached to a filter membrane through a ~ .

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dot-blot apparatus or Erom an agarose ~el by an electro-blotting technique. The DNA is ~etected directly by binding o anti- W -DNA antibody.
A quantitative analysis Eor DNA includes irradiating the sample containing DNA with ultraviolet light to form photoproducts. The DNA in the sample is then denatured and contacted with anti-UV-~NA antibody labelled with a siynalling means. The signal level thereby generated is compared with the signal level from a DNA sample having a similar G-C/A-T ratio containing a known concentration of DNA which has been subject to identical irradiation, denaturation and antihody treatment as the test sample DNA. This method, wilich can detect nucleic ~cids down to the picogram range, does not involve DNA-probes or other detection principles that clepend upon incorporation of radioisotopes or modified nucleotides.
Detection and/or quantification of total nucleic acid or total DNA accordin~ to the preset-t method may be used in recombinant DNA technology, such as in cDNA cloning, _ 20 where the amount of genetic material to be assayed is often too small for conventional agarose/ ethidium bromide visualization. The method may also be used to monitor the level of nucleic acid during the production of vaccines, hormones, monoclonal antibodies, and other pharmacological 2~ substances produced for in vivo administration. There exists a great need for demonstrating the absence of nucleic acid in such preparations, owing to the possible presence of oncogenes in contaminating nucleic acid. The present photoimmune detection system offers an easy, ~ inexpensive and sensitive method for achieving this goal.
A kit for quantifying total nucleic acid in a sample includes a supply of anti-nucleic acid antibody, a panel of nucleic acid-containing samples of known nucleic acid concen-tration as standards, signalling means Eor indicating the 36 binding of anti-nucleic acid antibody, and a means for com-paring the level of signal generated by nucleic acid in the test ~ample with the levels of signal generated by the ~58~

various members of the standard panel. The signalling means may suitably comprise any of the various si~nalling means discussed above, for example, an en~yme such as horseradish-peroxidase conjugated to a swine-anti-6 rabbit ~G, ancl su~strates Eor the enzyme providing a colorproducing reaction. The panel of standards ma-y be distributed in wells surrounding the test sample on a nucleic acid-binding support. The nucleic acid in the sample is qùanti-fied by matching the color intensity of tile developed sample with the appropriate standard.
The present method for detecting specific nucleic acid may likewise be adapted to kit form. Such a kit may contain a supply of UV-labelled nucleic acid probe molecules selected so as to hybridize with the nucleic acid to be detected. The ~it further contains a means for contacting the suspect sample with the probe molecules and a supply of anti-UV-nucleic acid antibodies which bind to the UV-labelled probe. The kit contains a signalling means for indicating the binding of anti- W -nucleic acid to the probe. The contacting means may comprise a support such as nitrocellulose paper onto which the sample nucleic acid is deposited.
The present method for photoimmune detection of specific nucleic acid may be used to demonstrate the 2~ presence of particular bacteria or other organisms; to determine,the presence of resistance and/or virulence-determinants in micro-organisms; to diagnose genetic disease to perform chromosome karyotype analyses; and to perform gene diagnostic methods such as oncogene analysis, tissue-typing or determination of predisposition to dis-ease. Each of the aforesaid procedures in~olves the detection and visualization of specific nucleotide sequences in genetic material under studyO
The present method of photoimmune detection of nucleic 3~ acids has the follo~ing noteworthy advantages over detecting means utilizing radioisotopes:

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(a) It is easy to label probes by UV irradia-tion. UV-lamps are standard equipment in most laboratories in the Eorn of ~ermicidal lamps. Even short probes, for instance, probes formed from synthe-tic DN~, may be utilized according to the present invention by first attaching a polypyrimidine nucleotide sequence to the 3'-OEI probe terminus. Such terminal sequences may contain many photo-products which can be easily recognized by antibodies.
(b) No expensive chemicals or costly radioactive nucleotides are required for probe labelling.
(c) The UV-labelled probe molecules are stable.
It is believed that such probe molecules may be stored for years after labelling. It is thus possible to generate and store extensive probe libraries for use accordin~ to the method of the present invention.
(d) Tne problems and dangers connected with handling of radioactive isotopes are avoided.
(e) The metilod is quicker ~han existing radio-isotope-labelling methods, as no time is spent in developing audioradiographic films, or in scintillographic counting.
Photoimmune detec~ion of nucleic acid has the following advantages over detection means utilizing biotinylated dUTP:
(a) Photoimmune detection, which relies on the generation of antigenic determinants in situ with W -light, is much simpler and quicker than assay methods using nick-translation for the incorporation of biotinylated dUTP.
b) The present method is more economical, as the use of expensive chemically-synthesized biotinylated dUTP is avoided.
(c) Labelling by nick-translation is unsuitable for extremely short probes, and is useless for probes formed from single-stranded nucleic acids. The present photoimmune detection method may be employed for all types of probes. Short probes may be used by first attaching a 35 polypyrimidine nucleotide sequence to the 3'-OH end of the probe.

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(d) The present method is at least as sensitive in detecting nucleotide sequences as the nick-translation biotin-labelling method.
(e) The present method may be used as a highly 6 sensitive technique to detect the presence o~ total nucleic acid in a sample, whereas the prior art methods may be used to detect specific nucleic acid sequences only.
The present invention is illustrated through the following non-limiting examples.
Example l Photoimmune Detection of DNA
Probes Hybridized To Sample DNA
Plasmid pPHR20 containing a 2.1 kb KpnI-fragment of ribosomal DNA isolated Erom the slime mold Physarum polycephalum was used as a probe. The probe DNA was labelled by UV-irradiation (254 nm, approximately 4 kJ/m2), linearized by cleavage with the restriction enzyme PstI and denatured by heat treatment (95C, S min.), followed by a quick chill in ice water to keep the DNA single-stranded.
The DNA samples to be tested were yenerated as follows~
The plasmid pPHR2n was cleaved into four fragments of the fo]lowing amounts with the restriction enzyme PstI:
(1) 250 ng; (2) 680 ng; (3) 68 ng; and (4) 6.8 ng. A
2~ fifth sampl~ comprised a 343 ng fragment of Kpn I-cleaved pPHR20. A sixth sample comprised 100 ng of the plasmid pPYAlOl. The fragments were separated by electrophoresis on , a 0.7% agarose gel. The gel was then soaked in alkaline solution ~0.2 M NaOH, 5 min.), neutralized in electrophoresis buffer, pH 7.5, ~nd transferred onto a New England Nuclear "Gene Screen" membrane by electroblotting (electrophoretic transfer of the DNA fragments out of the gel plane onto the attached membrane filter giving an "offprint" of the DNA fragment pattern on the membrane.) ~he membrane was then baked in a vacuum oven for 2 hrs at 80C. The UV-irradiated single-stranded DNA-probe was hybridized to the baked membrane at 42C for 18 hours in 50% formamide according to Maniatis, et al., Molecular Clonin~, p. 324-27. Following 362~

hybridization, nonspeci~ic antibody binding sites were saturated b~ incubating the membrane ~ith a(lO~5 solution of swine serum in tris-bu~Eered saline, TBS (20 mM Tris~HCl, pH 7.6, 0.5 M ~aCl) at 2~C for one hour with gentle shaking.
6 The primary antibody, rab5it-anti-UV-DNA antibody, in a 10% swine serum dilution, was subsequently allowed to attach to the membrane-bound DNA by incubation ~or 30 min at 20C. After washing (3x5 min. in TBS, 0.05% Tween 20) the secondary antibody, biotinylated goat-anti-rabbit IgG
was allowed to bind, followed by incubation with streptavidin and biotinylated alkaline phosphatase (protocol by the supplier - Bethesda Research Laboratory~.
The results shown in Fig. 3 illustrate the sensitivity of ~he 1etection system. The amount of DNA loaded in lanes l, 2 and 5 giving bands containing DNA in the lO0 ng range (which is the normal level for ethidium bromide staining), appears as heavily overloaded when probed with the photoimmune detection system. However, DNA bands in the O.l-lO ng range (lanes 3 and ~) are more _ 20 appropriate ~or qualitative identification.

Exam~le 2 Photoimmune Detection With DNA- ;
Probes Extended With PolydT-tails 25Photoimmune detection of specific DNA from the bacteriophage Hyl7 was demonstrated using DNA-probe mole-cules with appropriate poly-dT tail sequences. Hyl7 is an artificially-made bacteriophage consisting of a defined chromosome segment -from each of the bacteriophages P2 and P4.
Various quantities (8-lO00 ng) of the DNA-samples were applied to a membrane (Gene-Screen ~ybridization Transfer Membrane NEF-927, New England Nuclear) by means of a microfiltration apparatus (Bio-Dot Microfiltration Apparatus, Bio-Rad Labora-tories) in accordance with the instruction manual for the "Gene-Screen"membrane (New England Nuclear Catalogue No. NEF 872,Gene-Screen: Hybridization Transfer ~embrane-Instruction Manual).

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Single-stranded ("ss") and double-stranded (i'ds") DNA from phage lambda and single-stranded calf thymus DNA
("C.th~DNA"), were used as negative controls.
A pro~)e which consisted o Hyl7-DN~ cleaved wi~h the restriction enzyme HindlII was used for the hybridization-reaction. The fragments were provided with tails, on average of 1900 thymine-bases, by terminal transferase.
Prior to hybridization, the probe molecules were irradiated with 2500 J/m2 W -light (254 nm). The hybridization was performed in acordance with "~ethod III" in the afore-mentioned instruction manual for the "Gene-Screennmembrane.
After hybridization, the membrane was washed and incubated with 10% of normal swine serum in tris-buffered saline (TBS = 0.02 M Tis-HCl, ~H 7.6, with 0.5 ~ NaCl added) with gentle shaking at room temperatuer for one hour. This step was followed by incubation for 30 min. at 20C with a suitable dilution of anti-UV-DN~-antibody in TBS which contained 10~ swine serum. After washing ~3x5 min.) with (~ t r~ d ~
TBS containing 0.05'~ of Tween 20, the membrane was incubated _ 20 for 30 min. with swine-anti-rabbit-antibody which was linked to horseradish-peroxidase ~DAKOPATTS ~/S, DENMARKO) and diluted 1:200 in TBS with 10~ or normal s~ine serum. The membrane was washed again (3 x 5 min.) and incubated with diaminobenzidine and H22 according to Adams, J.C., Histochem Cytochem., 29: 775 (1981). The results appear in Figure 4.

From Fig. 4 it is clear that only the blots containing Hyl7-DNA, which is homologous to the probe-DNA, are stained. The specificity of the reaction is demonstrated by the absence oE a color reaction for ss and ds lambda-DNA, and for C.th-DNA. The stain furthest to the right contains only 8 ng of DNA, and thus provides an indication of the sensitivity of the technique. By increasing the sensitivity at the detection stage, it is beleived possible to detect 3 DNA in the pg-range.

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Photoimmune Detection of ~NA
Compared To Ethidium Bromide-Staining The sensitivity oE the present photoimmune a~ssay for detecting DNA was compared to the sensitivity of the conventional ethidium bromide-staininy technique according to the following ~rocedure~ DNA from P2, P4, lambda and pBR 322 were cleaved with the restriction enzymes HpaI, EcoRI, BgIII and HindIII, and separated by agarose-gel electrophoresis (0.7% agarose in the gel; 40 mM tris-acetate buffer with 1 mM EDTA, pH 8). The gel was stained with ethidium bromide (0.5 microgram/ml and visualized over UV-light from a transilluminator (Fotodyne, 300 nm) which simultaneously generated UV-photoproducts. The ethidium bromide-stained yel is shown in Fig. 5a. The DNA in the gel was denatured in 0.2 N NaOH and transferr~d to a "Gene-Screen" membrane by electroblotting according to the instruction manual Eor tile "Gene-Screen". The membrane was subsequently incubated with swine serum and stained according to the method described in Example 2. The result is shown in Fig~ Sb.

A comparison of Figures 5a and 5b reveals that photoimmune detection of DNA according to the present 26 invention (5b) works well, and is more sensitive than conventional ethidium bromide-staining (5a). For instance, in lane 2 from the top, showing P4 DNA fragmented with HpaI, several of the faint bands which can be seen in Fig.
5a are significantly stronger in Fig. 5b, thereby indicat-ing the increased sensitivity of the photoimmune detection over ethidium bromide-staining.

The present invention may be embodied in other specific forms without departing from the spirit or essential attri-butes thereof andl accordingl~l, reference should be made tothe appended claims, rather than to the foregoing specifica-tion, as indlcating the scope of the invention.

Claims (22)

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

1. A method for detecting the presence of specific nucleic acid in a sample comprising:
preparing a polynucleotide probe which will hybridize with the nucleic acid to be detected;
labelling said probe by inducing formation of UV-photoproducts by exposure to ultraviolet radiation, contacting said UV-labelled probe with substan-tially single-stranded nucleic acid from said sample under hybridizing conditions; and detecting hybrid complexes with anti-UV-nucleic acid antibodies labelled with a signalling means.

2. A method according to claim 1 wherein the polynucleotide probe includes a polypyrimidine nucleotide sequence at the 3'-OH terminus.

3. A method according to claim 2 wherein the poly-pyrimidine nucleotide sequence is generated by cloning said probe directly into a polypyrimidine nucleotide sequence.

4. A method according to claim 2 wherein the poly-pyrimidine nucleotide sequence is generated by polymerizing pyrimidine nucleotide molecules onto the 3'-OH terminus of said probe.

5. A method according to claim 1 wherein said anti-UV-nucleic acid antibodies are bound to the UV-labelled probe prior to contact with nucleic acid from the sample.

6. A method according to claim 1 wherein the signalling means comprises a color-indicating compound.

7. A method according to claim 1 wherein the signalling means comprises a reagent of high electron density.

8. A method acording to claim 1 wherein the signalling means comprises an enzyme that catalyzes a reaction forming a detectable reaction product.

9. A method according to claim 1 wherein a secondary antibody which binds to the anti-UV-nucleic acid antibody is labelled with the signalling means.

10. A method according to claim 9 wherein the signalling means comprise an enzyme that catalyzes a reaction forming a detectable product.

11. A method according to claim 10 wherein the enzyme catalyzes a color-developing reaction.

12. A method according to claim 7 wherein the enzyme is horseradish-peroxidase.

13. A method according to claim 1 wherein the sample nucleic acid is immobilized on a solid support.

14. A method for detecting the presence of specific nucleic acid in a sample comprising:
contacting single-stranded nucleic acid from said sample affixed to a solid support with a polynucleotide probe which will substantially hybridize with the nucleic acid to be detected under hybridizing conditions, said probe including a single-stranded polypyrimidine nucleotide sequence extending from its 3'-OH terminal end irradiated to cause the formation of UV-photoproducts;
incubating the support-bound nucleic acid with anti-UV-nucleic acid antibody to the UV-irradiated probe; and incubating the support-bound nucleic acid with secondary antibody which binds to said anti-UV-nucleic acid antibody, said secondary antibody being linked to an enzyme that catalyzes a reaction forming a detectable product.

15. A method according to claim 13 wherein said anti-UV-nucleic acid antibodies are bound to the UV-irradiated probe prior to incubation with the support-bound nucleic acid.

16. A method for detecting total nucleic acid in a sample comprising:
irradiating said sample with ultraviolet light to form UV-photoproducts in nucleic acid contained within the sample;
contacting the irradiated sample with anti-UV-nucleic acid antibody;
signalling the binding of said antibody to nucleic acid in the sample by a signalling means associated with said antibody.

17. A method according to claim 16 wherein the signal-ling means comprises a secondary antibody which binds anti-UV-nucleic acid antibody, said secondary antibody being linked to an enzyme that catalyzes a reaction forming a detectable reaction product.

18. A method for quantifying total DNA in a test sample comprising:
(a) irradiating the sample with ultraviolet light to form UV-photoproducts in DNA contained within the sample;
(b) denaturing the DNA in said sample;
(c) contacting the sample with anti-UV-DNA
antibody labelled with a signalling means; and (d) comparing the signal level with a signal from a DNA sample of similar G:C/A:T ratio containing a known concentration of DNA which has been subjected to identical irradiation, denaturation and antibody treatment as the test sample DNA in (a)-(c).

19. A kit for quantifying DNA in a test sample com-prising:
a supply of anti-UV-DNA antibody;
a panel of DNA-containing samples of known DNA
concentrations as standards;
signalling means for indicating the binding of anti-UV-DNA antibody to DNA in the test sample; and means for comparing the signal level generated by the DNA in said test sample with the signal levels generated by the various members of the standard panel.

20. A kit for detecting the presence of specific nucleic acid in a suspect sample comprising:
a supply of UV-labeled nucleic acid probe mole-cules containing UV-photoproducts, said probe selected so as to hybridize with the nucleic acid to be detected;
means for contacting the suspect sample with said probe to form hybrid complexes; and a supply of anti-UV-nucleic acid antibodies which bind said UV-labeled probe, said antibodies having a signalling means associated therewith.

21. A kit according to claim 20 wherein the associated signalling means comprises an enzyme-linked secondary anti-body which binds said anti-UV-nucleic acid antibodies.

22. A kit according to claim 21 wherein the probe includes a polypyrimidine nucleotide sequence at the 3'-OH terminus.