Method for ordering macromolecules by means of a moving meniscus, and uses thereof

2182q ~ 5 Wo 95/21939 ~ r ~. The present invention relates to a method of ~ nf ~ m ~ have 5 macromolecules such as polymers or macromolecules with biological activity, in particular DNA, or proteins.

The present invention also relates to the application of this method in methods of demonstration, measurement of intra mol ~ c ~ ire distance, separation and / or assay of ~ a macromolecule in an é ~ h ~ ntill- n Controlling the conformation of macromolecules represents an important industrial issue, for example in the manufacture of sensors or controlled molecular assemblies or in the problems of detection and analysis.

It can be interesting to have an elongated molecular conformation. By way of example, in the case where polymers are grafted onto a substrate, it has been proposed to extend them by the action of an electric field, of a flow or using optical tweezers.

In particular, in biology, DNA alignment - by electrophoresis (Zimmerman and Cox Nucl.

Acid Res. 22, p ~ 92, 1994), free flow (Parra and Windle, Nature Genetics, 5, p 17, 1993 and WO 93/22463) or in a gel (Sch ~ vartz et al.

Science 262, p 110, i993 and USP 33531) or using optical tweezers (Perl; ins et al., Science 26 ~ 1 p 819, 1994 and also USP 5079169) - opens up many possibilities in mapping, or in detection of pathogens.

These methods generally only allow an imperfect alignment, or even a transitory one - that is to say that the molecule is relaxed, once the stress has disappeared.

In the case of optical tweezers, the method is cumbersome, limited to a single molecule at a time, and difficult to carry out by unqualified personnel.

It has been proposed (1.

Parra and B.

Windle and WO 93/22463) a particular technique of DNA alignment by flow after cell lysis, then drying. The alignment obtained is very imperfect and inhomogeneous and numerous non-aligned clusters are observed.

The present invention relates to an original: -the and simple method for aligning macromolecules on the surface S of a support characterized in that one makes move on said surface S the line 21 ~ 2q (~ WO95 / 21939 r ~, l / rl ~ 5 16'i triple S / A / B (meniscus) resulting from the contact of a solvent A with the surface S and a medium B, said macromolecules having a part, in particular an end, anchored on the surface S, the other part, no ~ rll ~ the other end, being in solution in solvent A.

It has been observed according to the present invention, that the only passage of a meniscus on molecules of which a part is anchored on a substrate, the rest of the molecule being freely in solution allows them to be uniformly aligned perp ~ nriif ~ rement to the meniscus in movement leaving them adsorbed on the surface behind the meniscus.

This ph ~ nomi ~ is called here "molecular combing."

More p ~ r ~ lt ~ the stretching of the free part of the molecule is done by passing the triple line S / A / B, con ~ titl ~ nt the meniscus between the surface S, the solvent A and a medium B which can be a gas (usually air) or another solvent.

In a particular embodiment, the meniscus is a water-air meniscus, that is to say that the solvent A is an aqueous solution and the medium B is air.

In addition, it is possible to extend the air / water menchus used herein in order to stretch the molecule to other systems such as oil / water or water / surfactant / air, among others.

The displacement of the meniscus pcut be done by any relative displacement means of the fluids A and B with respect to the surface S.

In one embodiment, the surface S can be removed from the solvent A or conversely, the solvent A can be removed from the surface S.

In particular, the meniscus can be moved using mechanical means, pneumatic nf ~ t ~ mm ~ nt by sucking or blowing a gas, or hydraulic in particular by pushing or sucking the solvent A or the medium B.

Thus the displacement of the meniscus can be done by progressive evaporation of the solvent A.

When the meniscus is moved mechanically, it can be done either by translation of the A / B interface, or by translation of the surface S.

In a particular embodiment, the solvent is placed between two supports, at least one of which corresponds to said surface support S and the meniscus is displaced, for example by evaporation.

.. _ .. , . .. ,,, . , . , . , ..... , _ , _ , , _ _, _ _ _ ,, _ _ .

~ WO95 / 21939 21 ~ 9 ~ r ~ l ~ r ~ 5 ~ 6s Here, by ~ support ", any substrate whose cohesion is sufficient to resist the passage of the meniscus.

The support may consist at least on the surface of an organic or inorganic polymer, a metal, in particular gold, a metal oxide or sulphide, a semiconductor element or an oxide of a semiconductor element, such as 'a silicon oxide or a comhin ~ ic-m thereof, such as glass or ceramic.

Mention is more particularly made of ~ erre, silicon o, Yydé on the surface, graphite, mica and molybdenum sulphide.

As "support ~, one can use a single support such as a blade, beads, including polymer, but also any shapes such as bar, Rbre or structured support, and also particles, which s' acts of powders, in particular of silica powders, which can moreover be made fluorescent or colored magnetic as is known in the various n ~ (hnolo ~ ie.c assay.

The support is advantageously in the form of plates.

Preferably, the support exhibits little or no fluorescence.

Macromolecules, such as any polymers, or biological polymers such as DNA, RNA or proteins, can be anchored by 4uelcull4u methods on a support.

The macromolecule to be aligned can be chosen from biological macromolecules such as proteins, in particular antibodies, antigens, ligands or their receptors, nucleic acids, DNA, RNA or PNA, lipids, polysaccharides and their derivatives.

It has been observed, according to the present invention, that the stretching force acts locally in the immediate vicinity of the meniscus.

It is independent of the length of the molecule, of the number of molecules anchored, and in a wide range, of the speed of the meniscus.

These features are particularly interesting for aligning homogeneous and reproducible fason molecules.

One can, according to the present invention, add surfactant elements in the sol ~ ant A and / or ie medium B, which modify the properties of the interfaces.

According to the present invention the stretching can in crrct be controlled by the addition of surfactants, or by an adequate 3 ~ surrace treatment.

21 82 ~ 5 ~ r ~ l65 wo 95121939 Too much surface-macromolecule attraction (for example too high a level of adsorption) can freeze the alignment of the molecules by the meniscus, these remaining adsorbed on the surface in the a state not nec ~ di ~ ..t stretched.

Preferably, the surface has a low adsorption rate of said macromolecule, so that only the anchored molecules will be aligned, the others being carried along by the meniscus.

C.-rf ~ nl ~ nt, we can play on the adsorption differences between a part of the macromolecule rll ~ t ~ mm ~ nt its ends and its other 10 parts (in particular for long molecules, such as DNA or collagen) to anchor the molecules by adsorption by a part including their end (s) only, the rest of the molecule being freely in solution, on a very wide variety of surfaces and align them by passing the meniscus as described previously.

The adsorption of a macromolecule to a surface can be easily controlled using the pH or the ionic medium content of the medium or an electrical voltage applied to the surface.

The surface charges and the electrostatic interactions (repulsive or attractive) between the surface and 1 ~ molecule are thus changed, which makes it possible to go from a state of adsorp ~ complete ion of the molecule on 1 ~ surface to a total absence of 'adsorption.

Between these two extreme cases, there is a range of control parameters where the adsorption takes place preferentially by the ends of the molecules and which we will therefore use, with advantage, to anchor them to the surface, then to align them by the 25 passage of the meniscus.

The molecules, once aligned, adhere strongly to the suriace.

In the case of DNA, they could be observed by fluorescence, several months after their alignment.

The present invention is therefore very different from the method proposed by Parra and Windle, because according to the present invention, the molecules are anchored to the suriace and then uniformly aligned by the passage of the meniscus, whereas in the method of Parra and Windle a flow hydrodynamics is used to stretch inhomogeneously Ic ~ molecules q ~ ~ on ~ ads ~ r ~ er n ~ n ~ péci'quemen ~ ~ ~ a ~ urface.

~ Wo95J21939 ~ 1 82 ~ 0 ~ / r ~ i6 ~ Other techniques can also lead to the stretching and ipneml ~ nt of molecules.

Thus a dynamic orientation of molecules in solution anchored at one end can be obtained by electrophoresis or by hydraulic flow. However, the results observed 5 show that these techniqu es are much less efficient than the use of the meniscus.

By "anchoring ~ of the macromolecule on the surface is meant an attachment resulting from chemical reactivity both by a covalent bond and by a non-covalent bond such as a bond resulting from physicochemical interactions, such as adsorption as described above.

This anchoring of the macromolecule can be done directly on (or with) the surface, or indi ~ e ~ ..t, that is to say via a link such as another molecule, in particular a another molecule with biological activity.

When the anchoring is done indirectly, the macromolecule can be chemically grafted onto said link, or interact physicochemically with said link, in particular when said link in ~ c.iiai ~ is a molecule with activity biological l ~ n ~ t and interacting with said macrnmol ~ cl ~ le In one embodiment, the macromolecule and said link 20 are both molecules with biological activity which interact such as antigen and antibody resp ectively, complementary nucleic acids or lipids.

In these cases, the non-covalent attachment of the macromolecule consists of an antigen-antibody, ligandreceptor type binding, hybridization between complementary nucleic acid fragments or hydrophobic or hydrophilic interaction between lipids.

Advantage is thus taken of the very high specificity and the very high selectivity of certain biological reactions, in particular antigen / antibody reactions, DNA or RNA hybridization reactions, interprotein or avidin / streptavidin-type reactions. biotin, as well as the reactions of ligands and ~ eurs réCepteurS.

Thus, to achieve the direct or indirect anchoring of the macromolecule on the surface S, it is possible to use a solid surface having certain specificities.

It is in particular possible to use wogsl21939 ~ 1 82 ~ 05 ~ r ~ a ~ l65 certain surfaces p ~ C ~ .s pe ~ cult to ftxer certain proteins or DNA, whether or not it has been modified.

Such surfaces are commercially available (Covalink, Costar, Estapor, Bangs, Dynal for example) in various forms ~ ul ~ at their suface of ~ tC COOH, NH2 or OH for example.

The DNA can then be functionalized with a reactive group, for example amine, and a reaction can be carried out with these surfaces.

These methods, however, require a particular functionalization of the DNA to be fixed.

We have P ~ alPnnPnt describes a technique allowing anchoring without prior treatment of the DNA.

This process involves reacting a free phosphate from the 5 'end of the DNA molecule with a secondary amine from the surface (NH Covalink surface).

Anchoring by adsorption can be done by adsorption of the end of the molecule by controlling the surface charge using the pH, the ionic content of the medium or the application of an electrical voltage to the surface. given the differences in adsorption between the e ~ s of the molecule and its inte ~ part.

According to the present invention, it is thus anchored, by way of example, non-functi-lnn ~ lic ~ P ~ DNA molecules on surfaces covered with molecules terminated by a vinyl or amine group such as polylysine molecules or various surfaces such as glass, covered with molecules of the silane type terminated by vinyl or amine ~ l VU ~ lts or else 25 glass coverslips previously cleaned in an acid bath.

In the latter case, the surface of the glass in fact has SiOH groups.

In all of these cases, the pH range where the DNA is anchored is chosen to be between a state of complete adsorption and no adsorption, the latter being at a more basic pH.

It is understood that this technique is very general and can be extended by those skilled in the art to a very large number of types of surfaces.

One can also functionalize the DNA with a first reactive group or a P0 protein to make it react with a surface covered with a second reactive ~ u ~ elll, .lt or a 35 Pl protein, susceptible to react specifically with each other (or they) respectively, i.e. for example, P1 with P0.

The Po / PI couple can ~ W095 / 21939 21829 ~ r ~ l / r ~ a 165 be a couple of the biotin / streptavidin type (Zimmermann and Cox) or digoxdgenin / antibody directed against digoxigenin (anti-DiG), for example ( Smith et al., Science; ~, 1122 (1992)).

Preferably, the anchoring surfaces will have a low S rate of nu ~ this so as not to generate detection of molecules after their ~ nPmPrlt, in particular if this is done by fluorescence.

According to the present invention, use will preferably be made of a solid support having, under the reaction conditions, a surface having an affinity for part of the sP ~ IlPmPnt ~ macromolecule, the remainder of which remains freely in solution.

Dans.un embodiment, a solid support is used having on the surface at least one layer of an organic compound ~ S ~ dll ~, outside the layer, an exposed group having an affinity for a type of molecule to biological activity which may be said macromolecule itself or a molecule recognizing and / or interacting with it.

The support can therefore have a surface covered with a reactive group or with a molecule with biological activity.

By ~ affinity "is meant here both a chemical reactivity and adsorption of any type, this under the possible conditions of attachment of the molecules to the exposed group modified or not.

In one embodiment, the surface is substantially compact, i.e., it limits the access of the biologically active macromolecule to the lower layers and / or to the support, in order to minimize interactions. non-specific.

Surfaces coated with a reactive exposed moiety (eg, NH2, COOH, OH, CHO) or a biologically active macromolecule (eg: proteins, such as streptavidin or antibodies, nucleic acids) can also be used. such as oligonucleotides, lipids, polysaccharides and their derivatives) capable of binding an optionally modified part of the molecule.

Thus surfaces coated with streptavidin or an antibody according to known methods ("Chemistry of the protein Conlugation 35 and Cross-linking ~, SC

Wong, CRC Press (1991)) are capable of binding a macromolecule presenting at a particular site a biotin or an antigen.

21 8 ~ 90 ~ i WO gS / 21939 r ~, llrr ~ 165 ~ Similarly surfaces treated so as to present single-stranded oligonucleotides can be used to anchor DNA / RNA having a sequence ~ u ~ e.

Among the surfaces ~ u ~ u, uul L ~ ul ~ a ~. u ~ t reactive exposed, S we cite those on which the ~ IOu, u..ll ~ .lt exposed is a COOH group, -CHO, NH2, -OH, ûU a ~ IUUIJ..ll ~ .l ~ vinyl. UIIIUUII ~ t a double bond -CH = CH2 used as such or which can be activated to give nùl.,., .., 1 groups -CHO, -COOH, -NH2 or OH.

The highly specific surface supports according to the present invention can be obtained by carrying out various methods.

We can cite by way of example:

(A) a layer of carbonaceous polymer, optionally branched, at least 1 nm thick having ~ IOU, u .. The reagents as defined below and 15 (B) surfaces obtained by deposition or anchoring on a solid support of one or more molecular layers, these can be obtained by the formation of successive layers fixed by non- bonds. covalent, a type of non-limiting example, LangmuirBlodgett films, or by molecular self-~ cc ~ mhl ~ e, this allowing the formation of a layer fixed by covalent bond.

In the first case, the surface can be obtained by polymerization of at least one monomer generating the surface of the polymer said ~; IUU, U ~: lll..lt exposed, or by partial depolymerization of the surface of a polymer for generating said exposed group, or Z5 again by polymer deposition.

In this process, the polymer formed has vinyl bonds such as a polyenlque derivative, in particular synthetic rubber type surfaces, such as polybutadièn ~, polyisoprene or natural rubber.

In the second case, the h ~ utement specific surface comprises - on a support, a substantially monomolecular layer of an organic compound of elongated structure having at least:

a fixing group exhibiting an affinity for the support, and.

~ WO95121939 21 829155 r llrl 16 ~ an exposed group having little or no affinity for said support and said fixing group under the fixing conditions, but possibly having, after a chemical modification following the fixing, an affinity for a type of biological molecule.

The binding can first of all be of the non-covalent type, in particular of the hydrophilic / hydrophilic and hydrophobic / hydrophobic type, as in the Langmuir-Blodgett films (KB

Blodgett, J.

Am.

Chem.

Soc 57, 1007 (1935).

In this case, the exposed ~ 1uu ~ t or the fixing group will be either hydrophilic or hydrophobic, in particular alkyl or haloalkyl groups such as CH3, CF3, CHF3, CH ~ F, the other group being hydrophilic.

The binding can also be of the covalent type, the binding group will then react chemically on the support.

Certain surfaces of similar structure have already been mentioned in the electronic field, in particular when the bindings are covalent, L Netzer and ~.

Sagiv, J.

Am.

Chem.

Soc. 105, 674 (1983) and US-A-4,539,061.

Among the fixing groups, there should be mentioned more particularly groups of metal alkoxide or semiconductor type, for example silane, in particular chlorosilane, silanol, methoxy and ethoxysilane, silazane, as well as phosphate, hydroxy, hydrazide, hydrazine, amine groups. , amide, diazonium, pyridine, sulfate, sulfonic, carboxylic, boronic, halogen, acid halide, aldehyde.

In particular, as a fixing group, it will be preferred to use groups capable of reacting transversely with an equivalent, neighboring group, to provide the transverse bonds, for example these will be derivatives of metal alkoxide or semiconductor type, for example silane, especially dichlorosilane, trichlorosilane, dimethoxysilane or diethoxysilane and trimetho. ~ y or triethoxysilane.

The choice of the fixing group will obviously depend on the nature of the support, the silane-type ~ luu ~ ts are well suited for covalent fixation on glass and silica.

~ 1,829 ~ 5 W0 9S12193g ~ rl '' C 16 Regarding the gro-lrf-m.-nt. ~ EAposés, and whatever the surface, they will preferably be chosen from ethylenic ~ lUUp ~ S, acetylenic or aromatic radicals, primary, tertiary or se.ulldcu. ~ O amines, esters, nitriles, aldehydes, halogens.

But it could be very particularly the vinyl group; in fact, it can be either chemically modified after fiAation to lead, for example, to a carboxylic group or derivatives of ~; luu ~ l..ltS carboAyliques such as ~ luul,., - .. l ~ alcohols, aldehydes, ketones, acids, primary, secondary or tertiary amines, or lead to a direct pH-dependent anchoring of biological macromolecules such as nucleic acids and proteins without chemical modification of the surface or of the macromolecules.

Preferably, the chains connecting the eAposé group to the 1 ~ UU, U..Il..lt fixing are chains co ... pu. ~ UIt at least 1 carbon atom, preferably more than 6 and in general from 3 to 30 carbon atoms.

As regards the support itself, it is generally preferred to use glass, silicon oxidized on the surface, a polymer or gold with or without pretreatment of the surface.

One can have ~ r ~ "t use, in the case of glass or æ silica, the known techniques of surface functionalization using silane derivatives, for example: Si-OH + Cl3-Si-R-CH = CH2 gives Si-O-Si-R CH = CHz, R consisting for example of (CH 2) - ~ - Such a reaction is known in the literature, with the use of ultra-pure solvent. The reaction leads to a carpet of molecules exhibiting their end C = C to the surface 25 exposed to the outside.

In the case of gold, the latter optionally being in the form of a thin layer on a substrate, known surface functionalization techniques use thiol derivatives, for example: Au + HS-R-CH = CH2 gives Au-SR-CH = CH2, R consisting for example of (CH2) ~.

30 Such a reaction is described in a liquid medium and leads, like the previous trichlorosilane-silica reaction, to a carpet of molecules having their C = C end at the surface exposed to the outside.

~ WO95121939 ~ ir ~ llrl ~ 6s Of course the terminology of "support" includes both a single surface such as a blade, but ~ mPnt particles whether silica powder or polymer beads, and also any shapes such as bar, fiber or support structure, which can moreover be made magnetic, fluorescent or colored, as is known in dir ~ es tl ~ hnrllrlgi ~ c dosing.

From l, lr ~. ~ ..Ce, the support will be chosen to be little or no lluuL.O.elll when the detection is carried out by fluorescence.

The surfaces obtained according to modes (A) or (B) above have:

(i) a very low rate of intrinsic fluorescence, when required, a background fluorescence (with a typical area of ​​100 x 100 µm) lower than the fluorescence signal of a single molecule to be detected;

15 (ii) the possibility of detecting isolated molecules with an S / N ratio independent of the number of molecules, which is possible thanks to different high S / N ratio techniques described below and based on the identification of the presence of a macroscopic marker showing weak non-specific interaction with the surface.

The surfaces thus obtained are preferably coated with a macromolecule with biological activity chosen from:

- proteins, - nucleic acids, - lipids, - polysaccharides and their derivatives.

Among the proteins, mention should be made of antigens and antibodies, ligands, le ~ el.L ~ ul ~, but also products of the avidin or streptavidin type as well as derivatives of these compounds.

Among the RNAs and DNAs, mention should also be made of the derivatives ~, f, ~ insi as thio derivatives and mixed compounds such as PNAs.

It is also possible to add mixed compounds such as glycopeptides and lipopolysaccharides for example, or other elements such as viruses, cells in particular, or chemical compounds such as biotin.

21 8 ~ 9Q5 wogs / 21s3s ~ r ~ t ~ l6 12 The binding of biological macromolecules can be covalent or non-covalent, for example by adsorption, hydrogen bonds, ~ .ls h ~ ilopllobes, ionic, for example, in which case one can advantageously proceed with a bridging (Ucross-linking '') S between the molecules grafted by known methods (Chemistry of Prote in Conjugation and Cross-linking ", SC

Wong, CRC Press (1991)) and this in order to strengthen their cohesion.

As previously mentioned, it is possible to have an exposed ~ IOU ~, .t which allows direct reaction with the 10 molecules with biological activity, but it is also possible to provide that the exposed ~ u ~ lL is treated , after attachment, to be ~ rO.I .. ~, as has been indicated above, in a hydroxy radical, amine, alcohol, aldehyde, ketone, COOH or derivative of these groups before the attachment of the biological molecule.

iorsque such y, lOUI.) ~ III ~ III:> have been exposed, the protein and / or DNA binding techniques, for example, are known, they are indeed reactions implemented to surfaces which are already used in the context of biological analyzes, in particular for Costar surfaces and Nunc surfaces or microbeads such as Estapor, Bang and Dynai for example, on which molecules of biological interest, DNA, are anchored, RNA, PNA, proteins or antibodies for example.

In the case where the exposed group is a radicai -CH = CH2 which is named below ~ C = C surface ~ or "ethylenically bonded surface ~, there is no document m ~ ntionn ~ nt direct anchoring , in particular DNA or proteins.

In the context of the present invention, it has been demonstrated that these surfaces exhibit a strongly pH dependent reactivity.

This feature allows nucleic acids or proteins to be anchored using pH zones and often with a reaction rate which can be controlled by the pH.

The DNA can be anchored by its end on a surface preserltant ~ roupements has an ethylenic double bond by placing the DNA in the presence of the surface at a pH below 8.

in particular the reaction is carried out at a pH between 35 and ~ 5 and is then stopped at pH 8.

Wo95 / 21939 21 8 ~ 9 ~ / r ~ 165 1 13 Thus, for DNA at pH 5.5, the anchoring reaction is complete in one hour (if not limited by diffusion) and occurs by t :,. Llénli ~ t ~. At pH 8, on the other hand, I'd ~, is very low (reaction rate 5 to 6 orders of magnitude lower).

This hooking effect pH ~ l ~ r ~ nfl ~ nt and 5 specific ~ l elld ~ és, presents an improvement over other surfaces that require a ~ lic? Tior ~ DNA function (biotin, DIG, NHS, ...) or specific reagents tcarbodiimide, dimethyl pimelidate) which produce a peptide or phosphorimide bond between NHt and -COOH or -POOH.

It is also possible to achieve DNA anchoring by adsorption of its ends only on a surface covered with polylysine or a silane or ~ terminated with an amine group.

To anchor the DNA by its end on a surface covered by a ~. ~, U}, ~ .lt amine, the DNA is placed in the presence of the surface at pH between 8 and 10.

Likewise, DNA anchoring can be carried out by its end on a glass surface previously treated in an acid bath, by bringing the DNA into contact with said surface at a pH between 5 and 8.

It goes without saying that the present invention involves, in the same spirit, the attachment optionally pH depending on all macromolecules of biological interest.

Similarly, these surfaces can anchor dil ~ llt proteins (protein A, anti-DlG, antibody, streptavidin, etc.).

It was observed that (i) the activity of the molecule can be preserved and (ii) that the reactivity of the prepared surface (initially CC) is completely obscured to make way for the only reactivity of the molecule of interest.

It is therefore possible, from a relatively large initial reactivity, to switch to a surface having a very highly specific reactivity, for example that of specific sites on a protein.

By anchoring a specific antibody on the surface (for example anti-DlG), a surface is created whose reactivity is limited to the antigen (for example the DIG group).

This indicates that the initial chemical groups have all been occulted by the grafted antibodies.

2 1 82q ~ 5 ~ 16S Wo 95/21939 P ~ l / r 14 It is also possible to graft onto reactive surfaces (chemically or biochemically) other molecules with biological activity, nvL; -. ~ .Lt viruses or other components: membranes, lece ~ utt: u ...., membranes, polysaccharides, PNA, not: lmm ~ nt S 11 is IS ~ Pmrnt possible to fix the product of a reaction of biological interest (for example PCR ) on prepared surfaces.

The method according to the present invention allows the detection and / or the u ...- .t; li. ~ Linn of biological molecules, but also the measurement of intramolecular distance, the separation of certain biological molecules, l1u ~ .lt a sample by implementing antigen / antibody and / or DNA / RNA coupling techniques.

In particular, the present invention has for obje ~ a method for demonstrating a macromolecule consisting of a DNA sequence or a protein in a sample, according to the present invention, characterized in that:

- We put the sample ~ ul ~ OI, o. ~ dant ~ you solvent A, wherein said macromolecule is in solution, in contact with the surface of the support under conditions of formation of a DNA hybrid ~ DNA, DNA / RNA or of formation of the protein / protein reaction product, the hybrid or a labeling macromolecule of the hybrid or of the reaction product being anchored in one part, the remainder being free in solution, it is stretched by displacement of the meniscus created by the contact of the solvent with the surface in order to orient the hybrids or said labeling macromolecules and the measurement or observation of the hybrids or said labeling macromolecules thus oriented is carried out.

Advantageously, the fixed DNA and the DNA of the sample are colored differently and after stretching, the position of the complementary sequence is measured with respect to the end of the DNA of the sample.

Suitably the ELISA or FISH detection methods can be used.

The DNA sample can be the product or the substrate of an enzymatic amplification of DNA such as PCR, that is to say that the amplification of the DNA can be carried out once the latter anchored and aligned according to the method of the invention or before its anchoring and its ~ nempnt ~. ,. .. ... _. ..... ... _ _ _ ~ W095 / 21939 21 8 ~ 9 ~ rr ~ 5 The passage of the meniscus, by linearly stretching the molecules, in the form of rods, makes them more f ~ rilr-nnr-nt d ~ t ~ C ~ hlP ~ si they are marked.

Moreover, these elongated molecules are stable in the open air and can be observed even after several months, without showing any apparent degradation.

During rehydration, DNA molecules can remain adsorbed and elongated.

In addition, it is possible to carry out a hybridization on the elongated molecule.

In addition, exhibiting a correlated signal and of uniform orientation by virtue of their stretching, these molecules are distinct from the surrounding noise.

It is therefore easy to ignore the dust, the inhomogeneites, which do not exhibit any particular spatial correlation. Alignment is also interesting because in solution, the molecules in balls fluctuate thermally, which causes very large variations in their fluorescence signal, preferably collected with a shallow depth of field, and limits their observation.

The present invention therefore allows the observation of isolated molecules with a very high signal-to-noise ratio. (S / N).

It is remarkable that this ratio is independent of the number of molecules anchored.

The S / N ratio posed by the detection of a molecule is the same as for 10,000.

In addition, this stretching technique makes it possible to easily discriminate between molecules of various lengths.

We can ~ d .. ~ p ~ proceed to the following steps to further improve the S / N ratio:

- The molecule being stationary, we can integrate its fluorescence signal.

- The observation under the microscope presents a reduced field (typically 100 ~ mx 100 ~ m with an objective x 100 with immersion, NA = 1.25).

For a rrh ~ ntillon of 1 cm2 one can either carry out a scanning, or consider the use of smaller agr ~ nrli ~ r-Tn ~ nts objectives (x 10 or x 20) but high numerical aperture.

- The rods being always parallel, we can consider an optical spatial filtering method to further increase the S / N ratio.

21829 5 wo g5121939 D ~ l / r ~ .65 16 - Other overall fluorescence methods can be envisaged, such as those described in European patent application EP 103426.

- The linearization of molecules is observed both in the context of physicochemical anchoring and in the case of bonds of the immune type (DlG / anti-DlG).

- Once the surface is in the open air, the DNA molecules are stable (remain intact, even after several weeks) and fh ~ This property can advantageously be used to defer the anchoring step from the tracking step / count of anchored mol.6c ~ c, if this detection is done for example, but not limited to, by ~ o ~; e to lluule ~ ce.

Such use is covered by the present invention.

- A double (or multi) fluorescence technique can ev ~ ntl..ollPm ~ n ~ serv ~ r to improve the S / N ratio or to detect a double functionality.

The stretched molecules can be revealed by various enzymological or other methods, such as fluorescence or the use of hot or cold probes.

Their detection can be done by measuring a global signal (for example fluorescence) or by individual observation of the molecules by Illi.l usco ~, ie optical fluid ~ e ~ this ~ ie electronic, local probe methods ( SlM, AFM, etc.).

Thus generally, the present invention allows the demonstration, separation and / or assay of a molecule in a Prh ~ ntill ~ n, by a process ~ al, ~ .isé in that a surface is used capable of specifically binding said molecule, and in that the detection, separation or assay are carried out using a fluorescent reagent or not detecting the presence of the attached molecule.

Among the reagents, a distinction is made between fluorescent reagents and non-fluorescent reagents.

Fluorescent reagents contain molecules flu ~ JIescellle; ~, chosen with advantage to be long molecules of size greater than 0.1 ~ m and reacting speciflque directly or indirectly with the pretreated surfaces.

For example, but not limited to, a double-stranded DNA molecule stained using wogsnl93g 21 8 ~ 9 ~ 5 I ~ l / r ~ 6 ~ 17 probes flU ~ C ~ AAt ~ s ( ethidium bromide, YOY0, fluorescent nucleotides, etc.) which can be anchored di ~ lt by one or more eA ~ és on a surface ~, l; .. ~ .lldllL optionally a group of vinyl, amine or other type. ~ f., l by a judicious choice of the pH or of the ionic content of the medium or by application of an electrical voltage to the surface.

It is f ~ qlf ~ mf nt possible to use a particular functinnnqli ~ qtion of the molecule (DIG, biotin etc ...) to anchor it at one or more points on a surface having complementary sites 10 (anti-DlG , streptavidin, etc.).

The non-fluorescent reagents allowing the detection of molecules pr ~ qlqhl ~ mf ^ nt aligned according to the present invention can consist in particular of beads or microparticles anchored by means of another molecule specifically attached directly or indirectly to the molecule aligned and exhibiting only weak non-specific interaction with the surface.

For example, mention may be made of Dynal beads coated with streptavidin allowing anchoring on biotinylated DNA, aligned according to the present invention.

Depending on whether the desired molecule is detected directly by fluorescence or indi. ~ L ~ .ll.llt using the reagents above, we will talk about "direct detection ~ or by flag ~.

In order to limit the problems associated with excessively slow reaction times, one can advantageously reduce the diffusion times of the reagents to the surface by using small reaction volumes.

For example, but not limited to, by carrying out the reaction in a volume of a few microliters determined by the spacing between two surfaces, one of which is treated to present reactive sites and the other is inert or treated so as not to exhibit reactive sites, under the reaction conditions.

The detection of the number of aligned molecules can be carried out on a small number of molecules (typically 1 to 1000), by a low noise macroscopic physical test requiring neither electron microscope nor radioactivity nor nec ~ di ~ slow PCR 21 8290 ~ woss / 2ls3s r ~ llr ~ -l-i6s 18 The methods of ~ n ~ m ~ nt and detection according to the present invention are likely to be implemented by people with little laboratory experience.

The specificity of some biological reactions may be limited.

Thus, in the context of hybridization, the hybrids can be alra.ilO (reactions with other sites) while exhibiting a reduced number of pairings and therefore a lower quality of binding.

The present invention covers ~ g, ~ l..l. ~ .Ll the possible use of a step of testing the quality of the links obtained.

This test makes it possible to dissociate the paired products in a hible non-specific manner, by adsorption, hybrophobic forces, imperfect hydrogen bonds, hybridization i, .lpa. rdile, nr, t ~ mm ~ nt This is why the invention also relates to a method of detection or assay as described pr ~ 6r ~ mmrnt, a process where the reaction product is subjected between the molecule with biological activity and the molecule of the sample to a constraint in order to destroy the bad ones; I ~ IJdl i .. ts before detection.

This method offers, besides the possibility of destroying the couples s, the possibility of orienting the products of the coupling, which facilitates the measurements or the observations.

It is thus possible to apply to the surfaces, after fixing the elements ~ silent, a constraint which can be constituted by the single or combined use of:

- centrifugation, 2S - magnetic field gradient applied to non-fluorescent reagents then taken to include m ~ nétic ~ hles or magnetic microbeads, - stirring, - liquid flow, 30 - meniscus passage, - electrophoresis - temperature variation, and / or temperature gradient.

The low noise detection techniques described above are then determined to determine the number of systems which have remained intact or have been destroyed.

~ WO95 / 21939 ~ 1 ~ 29 ~ 5 r ~ llr ~ 6 ~ 19 The alignment and detection techniques described according to the present invention can be used for many applications among which, but not limited to:

- The identification of one or more elements of DNA or RNA sequence that can be used with advantage for the diagnosis of pathogens or the physical mapping of a genome.

In particular, the techniques described above make it possible to obtain a direct physical cd- ~ uK.dphie on genomic DNA, without going through a cloning step.

It is understood that the combed molecule being stretched with respect to its crystallographic length, relative measurements are taken.

It is thus possible to measure the size of DNA fragments and the distance between fragments with a resolution of the order of 200 nm for optical methods or of the order of 1 nm by the use of near-field methods such as than AFM or STM to visualize and measure the distance between probes on aligned DNA.

This naturally leads to:

1) the detection of deletions, additions or translocations of ~ 0 Kenomic sequences, in particular in the diagnosis of genetic diseases (eg Duchesne myopathy);

2) the identification of promoters of different genes by measuring the distance between the regulatory sequences and that awarded;

3) localization of regulatory proteins by identifying their position along DNA or the position of their target sequence;

1) partial or total sequencing by distance measurement using near-field microscopies (by e ~; example AFM or ST ~ I) between probes hybridized ap ~ al le.la ~ lt to a base of oligonucleotides of given length.

- Enzymatic amplification in situ on aligned DNAs.

- Improving the sensitivity of ELISA techniques with the possibility of detecting a small number (possibly less than 1000) of immunological reactions.

WO95 / 21939 ~ 1 829 ~ 5 l ~ llr ~ 16s Thus, it is possible to carry out a physical mapping directly on a genomic DNA without going through a cloning step.

Genomic DNA is extracted, purified, optionally cut with one or more restriction enzymes and then combed onto surfaces according to the method of the present invention.

The position and size of the gene retrieved on the genomic DNA are then determined by hybridization with specific probes for said gene, in particular extracted from parts of the complementary DNA (cDNA) of the product of said gene retrieved.

Similarly by hybridizing a combed genomic DNA, then denatured with total cDNA labeled by fluorescence or any other marker p ~ llt to locate the hybrid, we identify the position, size and number of exons of the gene in question , from which we deduce its size and its genetic gold ~ nic ~ tk-n (exons, introns, regulatory sequences).

The position of the gene being determined as described above, or known, it is then possible to identify by hybridization the flanking sequences of the gene.

For this, one proceeds, with advantage, by hybridization with labeled probes, originating by e, Yemple of an oligo- ~ ucl ~ otides library, to identify two or more probes which hybridize on both sides. other of the gene.

From this determination, it is then possible by enzymatic amplification techniques, for example in situ PCR (Nuovo GJ PCR in situ hybridization: ~ n. ~ L ~ .ul ~ and applications, Raven Press (lg9 )) to amplify the fragment delimited by the flanking probes which can serve as primers for the reaction, which fragment can contain the desired gene with its regulatory regions which can be tissue or specific development and which can then be isolated and purified .

It is also possible to proceed by in situ polymerization on primers extracted from the cDNA of the gene in question to extract DNA fragments complementary to the flanking regions of the gene as mentioned by Mortimer et al. (Yeast 5, 3-1 1989).

These fragments can then be used in the preparation of primers ~ s for a process of tmp ~ ii ~ ication enz ~ matique of the gene and its nanquantes sequences.

~ WO95121939 ~ 9 ~ 5 r ~ llr ~ c ~ -l6 ~ The methods mentioned by A.

Thierry and B.

Dujon (Nucl.

Acid Research 20 5625 (1992)) to insert by recomhin ~ icon homolog or al ~ law ~ cnt specific known endonuclease sites in genomic DNA or a fragment of genomic DNA.

The combing of this DNA allows the identification of the gene of interest and of the specific sites inserted, by the in situ hybridization methods described above. From this identification and preferably if the sites of interest are regions of interest close to the gene, they will be used as the primer for an enzymatic amplification reaction (in situ or other) of the gene in question and its sequences. flanking.

Amplification of the desired gene then proceeds by known enzymatic amplification techniques such as PCR on the fragment amplified as described above using primers accessible by the exons constituting the cDNA, or primers corresponding to flanking sequences.

By combing genomic or other DNA, it is also possible to determine by hybridization the presence or absence of regulatory sequences of a particular proximal gene, from which one will determine the possible families of regulatory proteins of this gene (e.g. example: helix-loop-helix, zinc-finger, leucine-zipper).

The specific reactions between particular DNA / RNA / PNA sequences and another molecule (DNA, RNA, protein) can take place before or after alignment of the molecules according to the present invention.

Thus, in the context of genetic diagnosis and physical mapping, the known methods of FISII (Pinl; el et al., Proc.

Nat.

Acad.

Sci. USA ~, 2931 (1986)) to hybridize labeled single-stranded oligonucleotides to DNA that is first aligned and then denatured.

The hybrids will be revealed by known techniques (fluorescences, microbeads, etc.) with a resolution in the measurement of distances ranging from 0.2 μm (in optics) to Inm (in near-field microscopy; AFM, STM, etc.). .).

Alternatively, one can first hybridize ~ r fluorescent marker DNAs to single-stranded DNA in solution, then align 35 this construct by the action of the meniscus after having transformed it into double-stranded DNA and anchored to a adequate surface.

~ 1 8 ~ 9Q5 WO 95/21939 ~ rn7. "'~ ~ 16S 22 The present invention can also be used for the detection of the presence of a pdlllG ~, _. Ie. By way of example, one can proceed in two different ways depending on whether the reaction of ~ o ~ nre (hybridization,. ~ ..t protein) takes place before or after ~ nFmFnt by the meniscus.

Thus, by way of example, one or more oligonurl ~ otiFlF ~ c probes are anchored 3. one or more regions of a surface.

The hybridization of the po ~ iFIl ~ "pathogenic Fnt DNA is carried out in situ under stringent conditions so as to anchor only the hybridized molecules.

Their detection and ~ IUdll ~ iri ~ d ~ iOn is eFfectuent after ~ nF ~ mF ~ nt by the meniscus according to the present invention.

Alternatively, the potentially pathogenic DNA is first aligned, then denatures and hybridized with an oli ~ ol ~ u ~ leotide probe in rr ~ nflitir ~ nc ~ llillhF ~ The detection of the hybrid is then carried out by the methods known in particular from FISH, as described above.

Similarly, one can detect the presence (or absence) of a small number of mr ~ c, such as proteins, lipids, sugars or antigens.

We proceed with advantage, to a mr, ~ lifir:? Ticn mlneure techniques of ELlSA, the usual detection method being replaced by the detection of ~ a fluorescent molecule aligned according to the present invention and coupled to one of the reagents of the ELISA reaction.

By the way, as mentioned by KR

Allan et al. (US 84,114), Genetic mapping can proceed by measuring the size of rld ~ d ~ DNA.

However, the original techniques for stretching the molecules described above (stretching through the meniscus) allow a measurement of the length of the stretched molecules and this on a very small sample (a few thousand molecules).

You can, for example, but without restricting it, proceed as follows:

A DNA sample is fragmented (using restriction enzymes), stained with a fluorophore and then anchored to a surface.

The molecules are then stretched by the meniscus and the size of the stretched fragments determined by fluorescence optical microscopy with a resolution and a size limit of the order of 1000 bp (0.3 ~ m).

WO95121939 21 ~ Q5 r ~ llr ~ 5 / c-l6s.

23 For this purpose, but also if we want to align very long molecules (2 lO ~, lm) use with advantage of known techniques to limit the degradation of long macromolecules during their handling (by hydrodynamic shear).

So as mentioned by DC

Schwartz, we will proceed with advantage, with a condensation of the molecules using a condensing agent (for example spermine or an alcohol) during their manipulation.

Optionally, their decondeDsation will take place during contact of solvent A with the anchoring surface S.

In order to reduce the degradation of the macromolecules during stretching by the meniscus, we will use techniques of translation of the meniscus which ~ lls-llt the fi ~ rnl ~ nt hydrodynamic.

For example, but without being limited to it, by withdrawing very slowly (s200 ~ / sec) the surface S, from a volume ffn ~ f ~ nt (2 100 ~ 1) of the solvent A.

A subject of the present invention is also a surface having one or more types of aligned macromolecules obtained according to the present invention.

In particular, one can obtain a surface or a stack of surfaces having electrical or optical properties anise ~ lo ~ c ~.

The present invention also relates to a method of ~ alignment and demonstration of DNA in which the DNA is stretched by an alignment method according to the invention, then denatured and then hybridized with specific probes to determine the position or size of one or more speciflc sequences.

A subject of the present invention is also a method for the physical mapping of a gene on a genomic DNA in which the DNA is aligned or demonstrated according to a method of the invention.

In particular, the position and the size of the desired gene on the genomic DNA are determined by hybridization with specific probes for said gene to be mapped.

The present invention also relates to -a box used for the implementation of a mapping method according to the invention, constituc p ~ r of the total genomic DNA of a reference host, Wo95 / 21939 ~ 1 8 ~ 9 ~ 5 r ~ llr ~ llol6s ~ 4 - a support having a surface allowing anchoring and ali ~ m ~ m ~ nl of the DNA of the patient in accordance with the method of the invention - specific probes of the or gene (s) to be mapped and reagents for hybridization and detection of DNA.

A subject of the present invention is also a method for aligning and demonstrating DNA in which the DNA is stretched then denatured and then hybridized with specific probes to determine the presence or absence of one or more sequences. of DNA in said aligned DNA.

The present invention allows the implementation of a method for diagnosing a pathology linked to the presence or absence of a DNA sequence specific to the pathology in which an alignment method according to the invention is used. .

A subject of the present invention is also a kit useful for implementing a diagnostic method according to the invention, characterized in that it comprises a support, the surface of which allows the anchoring and alignment of the DNA of the patient according to a method of the invention, specific probes for the gene involved in the pathology sought and reagents for hybridization and detection of DNA.

A subject of the present invention is also a kit which is useful for implementing a diagnostic method according to the invention, characterized in that it comprises a support the surface of which has specific probes for the gene involved in a pathology, in particular. Optionally labeled pathogenic DNA, aligned according to the method of the present invention and optionally denatured; the reagents to prepare and mark the DNA of the patient in ~ ue of its hybridization (for e ~; eg photobiotin, I; it of ~ nicl; -translalion "or" random priming ") and reagents for the DNA hybridization and detection according to the in situ hybridization techniques as described above.

It is understood that combed probes rclatives ~ different athogenes may be present on di ~ Férent supports or on the same support. The identification of the corresponding pathogen can be done after hybridization, either spatially (the different probes are ~ W095 / 21939 21 8 ~ 9 ~ r ~ Jlr ~ co ~; l6 ~ separated Sp ~ ri ~ PmPnt for example by photochemical anchoring a ~ ant their combing) or by a difference in the fluorescence spectrum of the different hybrids, resulting from a preliminary differential labeling of the probes.

S Finally, ~ a present invention relates to a method for preparing a gene in which the position of said syr gene of the genomic DNA aligned by the method according to the invention is identified using a probe specific for said gene is amplified by enzymatic amplification, in particular PCR in situ the sequence of said gene and optionally its flanking sequences.

The present invention therefore makes it possible to implement a method for replacing a gene in the genome of a eukaryotic cell by the targeted insertion of a foreign gene using a vector containing said foreign gene prepared according to gene preparation process above.

The targeted insertion can be carried out according to the techniques described in WO90 / 11354 by transfecting eukaryotic cells with a vector containing said foreign DNA to be inserted flanked by two genomic sequences which adjoin the desired insertion site in the recipient gene. The insertion DNA can comprise either a coding sequence or a regulatory sequence.

The flanking sequences are chosen so as to allow by homologous recombination, as the case may be, either the expression of the coding sequence of the insertion DNA under the control of the regulatory sequences of the recipient gene, or the expression of a ~ 5 coding sequence of the recipient gene under the control of the regulatory sequence of the insertion DNA.

The genomic genes and the cDNAs obtained using the method for localizing genes according to the invention can be inserted into pressure vectors capable of inserting into a prokaryotic, eukaryotic or viral host cell.

Protein polypeptides and derived peptides are included in the present invention.

The description which will follow is made with reference to the figures appended on which:

- Figure 1 schcmatizes the detection of a pathogen in a fluorescent DNA molecule by hybridization with an anchor molecule;

21 829Q ~ Wo 95/21939 A ~, I / r ~. 165 ~ 6 - Figure ~ hl ~ m ~ tic.o genetic mapping by extension of DNA and the use of a DNA marker;

- Figure 3 c ~ -hrm ~ ticr the detection of an immunological re ~ ction (ELISA) using a "flag" molecule: a fluorescent DNA 5 used as a reaction marker;

- Figure ~ is a fluorescence photomicrograph showing the extension of phage A DNA by the advancement of the meniscus, on the left we see DNA molecules in solution stretched by the evaporation flow parallel to the meniscus, at right of the DNA molecules in the open air after their stretching perpendicular to the meniscus;

- Figures 5 (a) and 5 (b) are fluorescence microphotographs showing, respectively, a DNA labeled with digogixenin (DIG) on a surface coated with anti-DlG and stretched by the meniscus, and, in control, an unlabeled DNA on an anti-DlG surface, the very high specificity of the surfaces and the absence of non-specific anchoring will be noted;

- Figure 6 shows the diagram of the spreading of DNA by passage of the meniscus. DNA in solution is anchored to a treated surface.

The DNA solution is covered with an untreated round coverslip;

- Figure 7 represents histograms of the length of DNA molecules ~ combed on glass surfaces:

a) coated with silane molecules terminated by a ~ 5 amine group, b) coated with polylysine, c ~ cleaned in an ox ~ gene water / sulfuric acid mixture.

- Figure 8 shows DNA molecules combed on glass surfaces covered with pol ~ lSsine.

Note that the molecules attached by their two e ~ hoppers form loops.

FIG. 9 represents YACs combed by removing a treated cover slip from a solution of these molecules.

- Figure L0 shows the identification of the presence e ~ the size of a cosmid on a YAC by in situ hybridization.

~ W095121939 21 ~ d? Q @, 5 1 ~, 1 / r ~ 165 Z7 In the "diagnostic ~ mode, the probes (the anchors) have a ~ U ~ reagent (DIG, biotin, etc.) capable of s' anchors} in a specific manner to a surface according to the present invention (having for example an anti-DLG antibody or streptavidin as an anchoring site).

The detection of the anchoring reaction can be done directly by detecting the llu ~ les; ~ of the DNA molecule stained by flUul ~ molecules (ethidium bromide, YOYO, nllc ~ ti ~ fluorescent) (Figure 1).

It can also be done ill ~ by detection of a "flag molecule: a reagent capable of binding to the DNA / RNA molecule (for example by hybridization, protein-DNA interaction, etc.), but not exhibiting d affinity for the anchor sites of the probe.

In the mode 'icartography ~ one can use the techniques of hybridization in situ (FISH).

It is also possible to envisage other techniques, for example by hybridizing DNA in solution with probes exhibiting fluorescent reagents according to the present invention.

The detection of the position of the probes is done after the ~ n ~ m ~ nt of the molecule according to the present invention.

EXAMPLE 1 Materials and Methods The DNA- and the monoclonal antibody (Anti-DlG) are from Boehringer-Mannheim.

The trichlorosilanes come from RothSochiel.

The fluorescent nucleic probes (YOYO1, YOY03 and POPO1) are from Molecular Probes.

The ultra-clean glass coverslips come from Erie Scientiflc (Lamelles (ESCO).

The magnetic particles come from Dynal.

The microscope is a Diapho t inverted microscope from NIKKON, equipped with a Xenon lamp for epiiluorescence and an intensified Hamamatsu CCD camera for visualization.

Surface treatment Glass coverslips are cleaned for one hour by UV irradiation under an oxygen atmosphere (by formation of ozone).

They are then imm ~ t ~ m ~ nt deposited in a desiccator previously purged of traces of water by a stream of argon A volume of about 100 to 500 ~ LI of the appropriate trichlorosilane (H2C = CH- (CH2) N- SiCI3 is introduced into.

WO95 / 21939 ~ 829 ~ 5 i ~ I ~ r ~ c.65 ~, 28 le d ~ si ~ rel-r, from which the surfaces are removed after about 12 hours (n = 6) or 1 hour (n = 1).

When leaving, the surfaces are clean and non-wetting.

The functional groups of these S double bond surfaces (H2C = CH-) can be transformed into ~; IVU ~ tS carboxyls (-COOH) by soaking the treated larnels, as described above, for ten minutes in a solution of 25 mg KMnO4, 750 mg Na lO4 in 1 L of water, then rinsing them three times in ultrapure water.

The thus functionalized coverslips can react with proteins.

A volume of 300 μl of an aqueous solution (20 ~ g / ml) of proteins (protein A, streptavidin, etc.) is deposited on a coverslip functionalized as a group (H2C = CH-).

This coverslip is incubated for about two hours at room temperature, then rinsed three times in ultrapure water.

The surfaces thus treated are clean and wetting.

Surfaces treated with protein A can then react with an antibody, for example anti-DLG, by incubation in a solution of 20 g / ml of antibody.

Furthermore, on surfaces having carbo ~ groups, one can graft uligolluclévvides having an amine end (-NH2), 20rv` ~ ll of a solution of MES (50 mM, pH 55), Carbodiimide (lmg / ml ) and 5il1 of oligo-amino (10 pmole / 140 ~ 11) are deposited on a carboxylated surface and incubated for about 8 hours at room temperature.

The coverslip is rinsed three times in NaOH (0.4 M) then four times in ultrapure water.

The thus prepared coverslips can hybridize DNAs complementary to the anchored oligonucleotide.

~ ncras ~ e of native DNA on double bond surface A drop of ~ ~ 11 of a solution of A [) N- ~ labeled by Quorescence (YOYOl, POPOl or YOYO3, but without particular tcrminaison) of variable concentration and in different buffers (total number of 30 molecules <107) is deposited on a pretreated coverslip (C = C) and covered with an untreated glass coverslip (diameter 18 mm). 1 ~ preparation is incubated for about 1 hour at room temperature in an atmosphere saturated with water vapor.

In a buffer of 0.0 ~ ~ 1 MES (pH = 5.5), we observe ~ e an almost general anchoring of DNA molecules.

On the other hand in WO9SJ21939 ~ 1,829 ~ 5 ~ r ~ 55.1 165 29 a buffer of 0.01 M Tris (pH = 8) there is ap ~ u ~ ducune anchor molecule (ratio> 106).

This dependence can allow the control of the activation / deactivation of surhces (vis-à-vis DNA) via the pH.

The action of the meniscus on the molecule is limited to the immediate vicinity thereof.

The part of the molecule in solution in front of the meniscus fluctuates freely and the part left stuck to the surface behind the meniscus is insensitive to a ~ I '' "& f" f., T direction of the meniscus.

The degree of extension of the molecule is therefore uniform and inf ~ pf ~ nf ~ nt of its size.

Alicnement and detection of the anchor By the action of the menisaue By transferring the previous preparation in a dry atmosphere, the solution, while evaporating, will stretch the DNA molecules, anchored to the surface, perpendicular to the mf nicqu ~ La capillary force on the DNA molecule (a few tens of picoNewtons) is indeed sufficient to stretch fr ~ mrl ~ tf-mf ~ nt the molecule (greater than the forces of entropic elasticity), but too weak to break the bond between the end of the molecule and the treated surface. The DNA being labeled by fluorescence, one observes indivi ~ m ~ nt and easily the stretched molecules (total length about 22 μm). The anchoring between the surface and the DNA being limited to the ends, it was also possible to stretch DNA of phage ~, YAC or E coli (total length greater than 400 ~ m).

This preparation of stretched DNA, lluo ~ D ~ cllts and in the open air is stable for several days and can be observed non-destructively, by epifluorescence (Nikkon Diaphot inverted microscope with x100 objective, ON: 1.25).

Ancra ~ e and specific detection By treating the surfaces as described precf ~ mm ~ nt with a specific monoclonal antibody, their specificity can be controlled very precisely.

Thus, we tested the specificity of anti-DLG treated surfaces vis-à-vis DNA- ~ hybridized with an oligonucleotide complementary to one of the Cos ends and having a digoxigenin group (DIG) and vis-à-vis d ' Unhybridized DNA.

In the first case, an almost general extension of the anchored molecules was observed by action of the meniscus.

In the second 21,829 ~ 5 W0 95121939 P ~ r ~. ~ A-16 ~ i case, only a few DNA molecules (<10) were observed anchored in the whole sample.

It is therefore estimated that the specificity of the method according to the invention is better than 106.

DNA- ~ were also hybridized with olif / ~ n ~ rl ~ tides 5 complementary to one of the COS ends and attached to carboxylated surfaces, as described above.

The hybridization c ~ nrliti ~ nc (pure water at 40C) were not ~ L ~ lt ~ o ~ because in ~) nriition ~ LCo thaute salinity) the fluorescence of the YOYO1 probes disappears and the hybridized DNAs cannot be seen.

The DNAs thus hybridized could also be aligned by passage of the meniscus.

Sensitivity of the de ~ ection In order to determine the sensitivity of the detection method for the extension of the meniscus, drops of 2.5 lll of a DNA solution were deposited on the double bond surfaces. 05 M MES (pH = 5.5) containing a total of 105.10 ~ and 1000 molecules. The anchoring and the lifinl ~ m ~ nt are carried out as described above.

The coverslips are then observed by epifluorescence microscopy, to determine the density of the combed molecules.

This corresponds well to that estimated: approximately 4-6 molecules of DNA per field of view (100 ~ lm x 100 Im) for a total of 105 molecules of DNA.

For the lowest concentration, about ten molecules extended by the action of the meniscus were observed.

This number is essentially limited by the large number of fields of view n ~ CcOO. ~ O to cover the entire sample (approximately 25,000), which makes manual research difficult, but can advantageously be carried out at ~ lt ~ matically or also with a weaker objective, but with a larger field.

In conclusion, the sensitivity of the method according to the invention allows detection and individual counting of less than 1000 DNA molecules.

Det ~ endance of the stretching on the surface treatment The histogram of the lengths of DNA-i ~, grafted on different surfaces, then aligned by passing the meniscus shows a well-defined peak, but different for the different surfaces .

Thus on surfaces covered with a silane ending in a vinyl group, the DNA is stretched to about 22 ~, -m (see above) for surfaces ~ wo 95 ~ 21939 21 82 905 J ~ I / r ~ '~ l6 ~ 31 siianized with a ~ IUU ~ IC ~ It amine (-NH2), the histogram shows a peak at 21, ~ -m (Fig. 7 (a)) and on clean glass at about 18.5 llm (hg.

7 (c)).

Stretching therefore depends on the surface treatment.

SE ~ pr ~ 2 pf ~ iPn ~ re of ADI molecules ~ on dl ~. ~. sutfaces DNA molecular combing has been observed on glass surfaces treated in different ways.

We play on the adsorption difference between the e. ~, Of the molecule and the rest of it.

By adsorbing positively charged polymers onto a glass surface adsorption of the negatively charged DNA molecules is enhanced, when this charge is large the DNA molecule is stuck along its entire length and the DNA molecule is stuck together. combing is impossible.

But it is possible to modify the charge of the polymers adsorbed on the glass by modifying the pH conditions, in fact, the positive charges are carried for example by NH 2 groups which pass to the NH 2 protonated state for a pH lower than pK of the corresponding base.

In basic pH, the charges disappear and the surface no longer attracts DNA.

By controlling the pH it was observed that the DNA molecules in solution went from a state where they are c ~ llcllt stuck to the surface to a ph ~ se intLI ulédi ~ where they are anchored only by their ends then at a phase where the surface no longer has any affinity for DNA.

In the intermediate phase combing mnl ~ c ~ ire is possible.

We studied surfaces covered with a silane terminated by 25 a ~ I ~ U ~ t N ~ 2 for which a total sticking is observed at pH <8, combing for 8.5 <pH <9.5.

The number of combed molecules is maximum at pH = 8.5 it is divided by 2 at pH = 9 and by 4 at pH = 9.S.

We also determined the relative extension on this surface which corresponds to 1.26 as can be seen on histogram 2 of figure 7 which represents histograms of the length of DNA molecules combed on glass surfaces:

a) coated with silane ending in an amine group, b) coated with polylysine, c) cleaned in a hydrogen peroxide / sulfuric acid mixture.

,, Zl ~ 29 ~ 5 Wog ~ / 21939 r ~ llrl ~ s,: i6s ~ 32 We have also looked at surfaces coated with polylysine which have similar adhesion characteristics in pH: combing range 8.5 and have a relative extension lowest: 1.08.

A typical example can be seen in Figure 8 which shows DNA molecules combed onto glass surfaces coated with polylysine.

We notice that the molecules attached by their two ends form loops.

Finally, the same behavior was found on glass surfaces freshly cleaned in an ox, vgenated water / concentrated sulfuric acid mixture.

These surfaces are very moldy and pollute quickly, however, a combing range between 5.5 c pH <7.4 was observed while the strong adsorption domain is located at pH = 4.5 The relative extension of the molecules corresponds to 1.12.

E ~ PL ~ Ali ~ nement uniform and directional YAC lllg of YAC previously dyed in its agarose block using a fluorescent probe YOYO1 is heated to 68C, agarased, then diluted in 10 ml of MES (50m ~ f pH S.5).

Two silanized yarns (C = C surfaces) are incubated for -I, Sh in this solution and then withdrawn at about 170 ~ m / sec.

The YAC molecules are all aligned parallel to the direction of cover slip retraction (Fig. 9). The integrity of the molecules thus aligned is better than by evaporation after deposition between two lamellae.

~ Ivbridization of a cosmid on a YAC t ~ ei ~ born A YAC tinted as described above is anchored on a surface C = C (e3; ~ be two lamellae) then aligned by the meniscus, during the evaporation of the solution .

The probes (cosmids) are labeled by inco} poration of a biotinylated nucleotide by the technique of "randon priming ~.

The labeled probes (100 ng) and 5 ~ g of sonic salmon sperm DNA (. = 500 bps) are purified by precipitation in Naacetate and ethanol, then denature in formamidc.

The combed YACs are denatured between ~ two slats with 1 ~ 0, d do solu ~ Qq den ~ ur ~ n ~ e (f ~ rm ~ mide 70 i, ~ ~ SSC) on a pl ~ that wos5l2l939 21 829Q ~ i P ~ llr ~ ~ -165 33 heating at 80C for 3 minutes.

The denatured probes (20 ng) prr ~ l ~ hl ~ m ~ nt are deposited on the coverslip in a hybridization solution (55% formamide, 2 x SSC, 10% dextran sulfate) covered with a coverslip which is sealed with liquid rubber (rubber cement). Hybridization 5 is carried out overnight at 37 ° C. in a humid chamber.

The hybrids are revealed according to the protocols known for in situ hybridizations on chrrlmos ~ mr-c drrrnfll ~ c ~ c (D.

Pinkel et al., PNAS USA 83, Z931 (1986) and PNAS USA ~, 9138 (1988)).

Under fluorescence microscopy, hybridized segments such as the one shown in FIG. 10.

This example demonstrates the possibility of detecting the presence of a gene on a DNA molecule, which can be used for diagnostic purposes or for physical mapping of the genome.

.