Probe Containing a Modified Nucleic Acid, Recognizable by Specific Antibodies and Use of This Probe and Theses Specific Antibodies to Detect and Characterize a Homologous DNA Sequence

Additional features of the invention will also appear in the course of the description which follows of a typical example of the practising of the process.

Use is made of the following materials and methods.

Phage .lambda. DNAs come Biolabs Inc. (New England).

The ribosomic ribonucleic acid comes from beef liver and the reagents for the demonstration with alkaline phosphatase (RR Fast Blue, Naphtol AS-MX phosphate salt) come from the Sigma Firm.

The nitrocellulose filters, of BA-85 type come from Schleicher and Schull. The Staph A cells fixed by formalin (marketed under the name Immunoprecipitin) come from Bethesda Research Laboratories.

The "nick-translation" kits (ref. N-5 000) and the .alpha.-.sup.32 radioactively marked p-dCTP nucleotides, 800 ci/mmole) come from Amersham.

The AAF and the AAIF are synthesized according to the articles which appear in Biochemistry 15.3 347-3 351 (FUCHS et al.) and Biochemistry. 17. 2 561-2 567 (LEFEVRE et al). They are preserved in tubes wrapped in aluminum foils, under nitrogen at -20.degree. C.

The anti DNA-AAF and anti Guo-AAF antibodies are obtained as described in Febs Lett. 92, 207-210 (LENG et al).). Biochemistry, 18. 1 328-1 332 (SAGE et al) and in Nucleic Acids Res.. 6, 733-744 (GUIGUES et al).

The antibodies bound to peroxidase and alkaline phosphatase come from Miles Laboratories and Institut Pasteur.

The plasmids and M13 clones with the dzeta human globin cellular DNA inserts come from the INSERM U91 unit Henri Mondor Hospital, Creteil, France and from the Department of Medicine of the Yale University of Medicine (New Haven-United States). The 4p7-7 bears a cellular DNA fragment of 464 pairs of dzeta globin bases inserted in the pBR 322 at the Pst 1 site. The same fragment is inserted into the M13 (DNA 1 355-363, COHEN-SOLAL et al).

The monocatenary M13 DNA is prepared according to Messing et al in Nucleic Acids Res.. 9. 309-321.

The PWE6 clone comes from the Centre de Recherches de Biochimie et de Genetiue Cellulaire du CNRS, Toulouse, France. It contains a 45 S mouse ribosomic DNA of 6.6 kilobases inserted in pBR 322 at the EcoRI site (Nucleic Acid Res.. 10, 5 273-5 283. MICHOT et al. and Nucleic Acids Res.. 11, 3 375-3 391, MICHOT et al).

The nucleic acids to be modified are reduced into fragments of about 1,000 pairs of bases (pb) by sonication and dissolved in a sodium citrate buffer (12 mM, pH 7) at a concentration of about 500 .mu.g/ml. After denaturation by heating (100.degree. C., 5 minutes) and rapid cooling in ice, 1/10th of a volume of an AAIF solution in ethanol is added. The total amount of AAIF added must be equal to about 3 times the weight of nucleic acids to be modified. The mixture is incubated 3 hours at 37.degree. C., sheltered from light. After incubation, the excess AAIF is removed by three extractions with cold ethyl ether and preparation is treated with a borate buffer (50 mM, pH 9) at 100.degree. C. for 3 minutes, this in order to remove the inter-chain linkages which could have been produced. After neutralization with a Tris HCl buffer (100 mM, pH 7) the preparation is ready for use for the hybridization. It can be preserved at +4.degree. C., or preferably at -20.degree. C., for several years.

When radioactive DNA is used, the "nick-translated" DNA is precipitated with ethanol redissolved in citrate buffer and denatured by heat. Aliquot parts are treated as indicated above omitting the sonication step. The control DNA is treated with pure ethanol. After treatment, EDTA is added to a final molarity of 2 mM and the modified nucleic acids are preserved protected from light at 4.degree. C.

Procedure is as indicated in (1.degree.) without carrying out a sonication step.

The percentage of modified bases is determined by measuring the absorption at 310 nm and 260 nm as described in Biochemistry, 11, 2 659-2 666, FUCHS et al and Febs Lett. 34, 295-298 FUCHS et al. When this photometric method cannot be used by reason of specimens being too small, there are deposited on a nitrocellulose filter several dilutions of samples to be tested and a sample already measured. After immunochemical dyeing, the color intensities of the spots are compared.

The tests are done on DNA adsorbed on nitrocellulose filters by current techniques (filtration by means of an apparatus of the "Hybridot" type marketed by Bethesda Research Laboratories or transfer by capillarity by the method of Southern, described in J. Mol. Biol., 98, p. 503, 1975, but the use of other supports can be envisaged (for example nylon filters of the "Biodyne" type marketed by PALL, DBM (diazobenzyloxymethyl) paper (Cell, vol. 5, p. 301, Noyes and Stark, 1975 and Alwine et al. 1977, PNAS, 1974, p. 5 350, Kemp coll.).

The filters are prehybridized for 2 hours at 65.degree. C. in a solution containing:

The hybridization with the modified probe (DNA-AAIF or RNA-AAIF) is done at 65.degree. C. for the desired time in the following solution:

The hybridization time depends on the concentration of the probe and the complexity of the sequence which is sought. It can be calculated by known methods. The use of products accelerating the renaturation of the nucleic acids (for example dextran sulfate) or lowering the hybridization temperature (for example formamide) is possible.

After hybridization, the filters are rinsed to remove the excess probe following procedures currently used and described in the literature.

The hybridized filters are subjected to the following treatments:

saturation in proteins by incubation for one hour, at room temperature in a solution containing:

(saturating solution);

Placed in the presence of the first antibody (antibody recognizing the AAIF) for one hour at room temperature. This antibody is diluted with the saturating solution. The dilution ratio depends on the antibody used;

rinsing in a solution containing:

(rinsing solution):

incubation for one hour at room temperature with the second antibody, recognizing the first antibody (if the first antibody is a rabbit antibody, the second antibody may be, for example, a rabbit anti IgG antibody bounded to alkaline phosphatase.)

The second antibody is diluted in the saturating solution, in a ratio which depends on the antibody used.

rinsing by using the same rinsing solution as that indicated above;

development of second antibody by known techniques (for example colored enzymatic reaction of the alkaline phosphatase).

The tests were done by using as a target viral DNA (.lambda. phage) or human genes (foetal dzeta globin,460 pb) or mouse (ribosomic DNA 6.6 kb) cloned in bacterial plasmids (pBR 322) and as bicatenary DNA probe (DNA of .lambda. phage or pBR 322 plasmid), monocatenary (M13 phage DNA bearing a 464 pb insert of human dzeta globin), or RNA (beef ribosomic RNA).

For the detection of the probes, the first antibody was a rabbit anti-guanosine-AAF antibody (crude serum) at a dilution of 1/200 and the second antibody, a rabbit anti-IgG antibody bonded to alkaline phosphatase and used at a dilution of 1/400.

Under these conditions, the sensitivities obtained were as follows;

for bicatenary DNA probes used at a concentration higher than or equal to 500 ng/ml (500.times.10.sup.-9 g/ml): sensitivity better than 2 pg of target DNA (2.times.10.sup.-12 g);

for monocatenary DNA probes used at a concentration higher than or equal to 100 ng/ml: sensitivity better than 2 pg of target DNA;

for ribosomic RNA probes at a concentration of 200 ng/ml: sensitivity in the neighborhood of 200 pg.

It is recalled that a sensitivity of 2 pg of a target DNA corresponds to the detection of a gene of 1,000 pb in 7 .mu.g of total human DNA. This sensitivity is of the same order of magnitude as that required for prenatal diagnosis of falciform anemia.

Ribo- or deoxyribonucleic acids react easily in vitro at a pH with AAF and AAIF. Under the above described conditions, the derivatives are linked principally to the carbon 8 of the guanine residue by a covalent linkage. The antibodies obtained by immunizing rabbits with a DNA-AAF and Guo-AAF specifically recognize DNA modified by AAF. They recognize also DNA and RNA modified by AAIF. Results of table I below show a larger amount of DNA found in a precipitate when DNA is modified by AAIF.

Small amounts of normal DNA are precipitated by anti-DNA-AAF antibodies. To differentiate the modified or unmodified DNA, anti-Guo-AAF antibodies are preferably used since the results obtained are better. In fact, only negligible amounts of unmodified DNA are precipitated by purified anti-Guo-AAF antibodies. This permits the use of the probes for the separation of specific gene sequences from complex mixtures.

The detection of the modified nucleic acids by nucleic chemical techniques is done in the following manner. The modified DNA is bonded to the nitrocellulose filters and the filters are then treated as indicated above. Considering that the number of parameters to be studied is very large, small round filters, 25 mm in diameter are chosen since they are easy to manipulate. By using DNA modified by AAIF (5% modified base), the sensitivity limit is less than 1 pg when the second antibodies are linked to alkaline phosphatase and about 8 pg when they are linked to peroxidase.

As regards the DNA fusion temperature, it is approximately reduced by 1.1.degree. C. and 0.4.degree. C. respectively for 1% of base modified by AAF and AAIF (Biochemistry, 15, 3 347-3 351, FUCHS and coll.: Febs Lett., 34, 295-298, FUCHS et coll.; Biochemistry, 6, 117-182, KRIEK et coll.; Biochem. Biophys. Acta, 232, 436-450, KAPULER et coll.).

Hybridizations by spots were done to evaluate the effect of modifications of the AAF and of the AAIF on bicatenary DNA probes. Nitrocellulose filters with three DNA spots of PBR 322 (422 pg, 2 ng, 20 ng) and one spot of DNA (1 ng) were hybridized with radio-active DNA probes, either unmodified, or with about 5M of base modified by AAF or AAIF. After hybridization, given groups of filters were washed with more or less vigor, dried and auto-radiographed for 8 hours. No non-specific hybridization could be observed, even with the gentlest washing (NaCl/sodium citrate, 15 minutes at room temperature).

The stability of the hybrid is tested on filters with DNA spots (10 pg, 100 pg, 1 ng, 10 ng) which are washed with different forces after hybridization:

either with radioactive modified DNA probes;

or with controlled probes.

At each degree of washing force, a group of filters was dried and they were auto-radiographed. No appreciable difference could be seen between the three probes. No spot was observed on the filters hybridized with a control DNA probe whatever the washing force. The spot corresponding to 10 ng is slightly visible on the filter hybridized with a probe modified with AAF. With a probe modified with AAIF, the corresponding spot at 1 ng is also visible.

For each of the above-indicated experiments, each of the probe concentrations was 2 ng/ml, the specific activity was 5.times.10.sup.7 cpm/.mu.g and the auto-radiograph time was 18 hours. Under these conditions, the sensitivity of detection obtained with the probes modified by AAF and AAIF are about 1/100 and 1/10 compared with that obtained by autoradiography.

Considering that any type of nucleic acid can be modified either by AAF or AAIF, the possibility has been tested of using M13 monocatenary probes and immunocleic RNA probes in hybridization experiments. For hybridization with monocatenary M13 DNA, each filter contains a DNA control spot of phage .lambda. and decreasing amounts of DNA of plasmid 4p7- 7. The monocatenary DNA of M 13 recombinant phage with the same insert of 464 pairs of bases of dzeta globine is modified by AAF or AAIF (5% of pairs of modified bases). Several concentrations of a probe of 125 to 1 500 ng/ml were tested. Hybridization and washing were carried out at 65.degree. C. In this case, the concentration of the hybridizable sequences of the probe was about 33 ng/ml and the target sequences on the spots were comprised between 660 and 5 ng. All the spots were colored except the negative control. The coloration of the spots is more intense with the probe modified by AAIF.

Similar tests were done with RNA probes modified by AAIF.

The liver ribosomic RNA (Sigma, reference R 5 502) is dissolved in citrate buffer (2 mM, pH 6.7) sonicated briefly to reduce its size to fragments of about 1000 bases and modified by AAIF. Spots were formed with the DNA of pBR 322 (100 ng) to have a negative control, and decreasing amounts of DNA of pWE6, a recombinant plasmid with a ribosomic 45 S mouse DNA insert of 6.6 kb. The amount of sequences hybridizable on the spots is comprised between 30 ng and 230 pg. Different probe concentrations were used ranging from 200 to 2300 ng/ml. Only slight differences in the intensities of coloration were observed with higher probe concentrations. When immunonucleic probes are used in high concentrations, the background noise of the filters remains very low. The possibility of using high concentrations of probe is interesting when it is desired to make short hybridizations.

From all these results, it appears that the properties of the nucleic acids modified by AAIF renders them suitable for the detection of specific sequences.

The use of the iodized AAIF derivative in place of AAF contributes a ten fold gain in sensitivity.

In addition, when the second antibodies used are bonded to alkaline phosphatase, the sensitivity is in the field of picogram.

The invention is obviously not limited to the embodiments described above by way of example and technicians skilled in the art can introduce therein modifications without however departing from the scope of the following claims.

By way of modifications usable at the level of the detection of the hybrids formed with the probe according to the invention, is mentioned:

the development (by radioactivity) of the hybrids formed, by example by means of the use of anti-DNA-AAF antibodies rendered radio-active by iodine 125 or 131 or radio-active protein A, which will be fixed to the antibodies.

Finally, by way of modifications of possible uses, will be mentioned the application of the probe according to the invention to the purification of complementary DNA contained in an initial composition, particularly by means:

of protein A associated with a solid support (for example constituted by agarose beads);

precipitating antibodies associated or not with a solid support (agarose or latex beads, etc.); to ensure the selective precipitation of the hybrid formed.

The invention naturally thus extends its effects to any probe marked by modified groups, equivalent to the N-2-acetylamino-7-iodofluorene groups whose formula is recalled below: ##STR1##

In particular, equivalents of these marker groups are constituted by any groups in which the iodine atom at the 7 position is replaced by another atom or substituent group of several atoms, which, like iodine renders impossible the insertion of the fluorene nucleus which is then associated with it between two pairs of bases. Such a substituent group is for example constituted by an alkyl radical, particularly methyl or preferably tert-butyl. A peptide group can also be envisaged. Substitution on the fluorene nucleus can also possibly take place on another position of the nucleus, for example at the 9 position; this amounts, for example, to double methylation of the 9 position.

The introduction of other substituent groups on the fluorene groups or the modification of the fluorene groups by substituents which do not modify the essential immunogenic properties, can only lead to the constitution of equivalents, when the thus modified DNA probes are still recognized by antibodies previously formed against N-2-(guanosine)-8-yl)acetylaminofluorene or against molecules resulting from modifications of the latter group. These modifications must not however spoil their solubility in aqueous solutions, to the extent necessary for the production of antibodies against this latter type of molecule. In known manner this immunization is generally produced after coupling of the molecule concerned with a carrier molecule contributing to the reinforcement of its immunogenicity, for example a serum albumin. By way of other examples of modifications which cannot spoil the solubility, will be mentioned, for example, replacement of the N-2-acetyl group by an N-2-formyl group or N-2-propionyl group in the N-2-(guanosine-8-yl)-2-acetylaminofluorene.