WO2005014853A1 - Comparative expression analysis for the identification of genes responsible for phenotypic differences - Google Patents

Abstract

Described is a modified substractive hybridization assay and uses thereof. In particular, methods of triggering gene expression and subsequently identifying and isolating nucleic acid sequences are provided, wherein said nucleic acid sequences are unique for a certain cell, tissue or organism, and preferably related to genes that cause a phenotypical difference. The presented methods are particularly useful for the identification of novel genes involved in the development or prevention of various diseases, including cancer, hypertension and diabetes as well as for monitoring animals and food, for example for desirable breeding traits, infection and contaminants.

Description

Comparative Expression Analysis for the Identification of Genes Responsible for Phenotypic Differences

Field of the invention

The present invention relates to a modified subtractive hybridization assay and uses thereof. In particular, the present invention relates to methods of triggering gene expression and subsequently identifying and isolating nucleic acid sequences, which are unique for a certain cell, tissue or organism, wherein said unique nucleic acid sequences are preferably related to genes that cause a phenotypical difference. The presented methods are particular useful for the identification of novel genes involved in the development of various diseases, including cancer, hypertension and diabetes as well as for monitoring animals and food, for example for desirable breeding traits, infection and contaminants.

Background Art

The current automated sequencing technology allows the collection of genetic information in large scale. It is no longer a problem to sequence the whole genome of an organism. The challenge lies now in the correlation of the identified genes with certain phenotypes. Such correlation can be found by comparing the expression profiles of cells displaying the phenotype to be investigated with the profile of control cells, which do not display it. One approach for the comparison of expression profiles is the hybridization of arrays. Arrays of different formats are available commercially for different organisms or can be readily produced. But array hybridization usually leads to large numbers of genes that appear to be differentially expressed. In order to obtain genes potentially correlating with the phenotypic trait that can be investigated further one has to reduce the number. This is usually done by employing statistical methods which in turn requires a large number of samples to be profiled. Expression profiling by array hybridization is therefore costly and applicable only where sufficient sample numbers can be obtained. Another disadvantage is that only genes that are at least partially known can be assayed on an array. The identification of previously unknown genes is not possible. Another approach for the comparison of two expression profiles is subtractive hybridization. This process was commonly used in association with cloning of cDNA derived from mRNA extracted from particular cells that are under investigation and is most useful for producing DNA hybridization probes that can be utilized as screening agents to detect or locate DNA, in for example cDNA libraries. Genes differentially expressed as compared with genes of other cells that exhibit a different phenotype can be cloned this way even if their sequence is completely unknown.

This technique may, for example, be used in cancer research for comparing the gene products of tumor tissue cells with those of corresponding normal tissue cells in order to study the genetic changes that have occurred at the nucleic acid level. Probes obtained using this technique which are specific to DNA whose expression characteristics are modified by such genetic changes may be useful also as diagnostic tools. In a typical procedure for applying this technique of subtractive hybridization to investigate differences in the active genes of a certain sample of test or target cells, e.g. from tumor tissues, as compared with the active genes of a sample of reference cells, e.g. cells from corresponding normal tissue, mRNA is extracted using conventional methods from both samples of cells. The mRNA in the extract from the test or target cells is then used in a conventional manner to synthesize corresponding single stranded cDNA, the template mRNA finally being degraded by alkaline hydrolysis to leave only the single stranded cDNA. The single stranded cDNA thus derived from the mRNA expressed by the test or target cells is then mixed under hybridizing conditions with an excess quantity of the mRNA extract from the reference (normal) cells. The latter is herein generally termed the subtractive hybridization "driver" since it is this m-RNA or other single stranded nucleic acid present in excess which "drives" the subtraction process. As a result, cDNA strands having common complementary sequences anneal with the mRNA strands to form mRNA/cDNA duplexes and are thus subtracted from the single stranded species present. The only single stranded DNA remaining is then the unique cDNA that is derived specifically from the mRNA produced by genes which are expressed solely by the test or target cells. To complete the subtraction process the common mRNA/cDNA duplexes are then physically separated out using, for example, hydroxyapatite (HAP) or, more preferably, (strept)avidin- biotin in a chromatographic separation method, see e.g. Kwon. et al, Proc. Natl. Acad. Sci. USA 48 (1987), 2896-2900 and Sive and St John, Nucl. Acid Res. 22 (1988), 10937). The main difficulty of the technique is the complete removal of the mRNA/cDNA duplexes. Left over duplexes will cause a background of genes expressed in the test as well as in the control sample. Disadvantages of the usually employed separation techniques are the loss of a significant number of potentially interesting genes and the often incomplete separation leading to a high background caused by a significant number of false positive genes. Since this step is crucial various improvements of the subtractive hybridization method tackle the separation of duplexes.

For example a method of chemical cross-linking subtractive hybridization (see Hampson et al, Nucleic Acids Res. 20 (1992), 2899) was developed, which avoids the need for physical separation of the common mRNA/cDNA duplexes from single stranded unique cDNA. The duplexes will not serve as a template in the PCR amplification that follows chemical cross- linking. Therefore only the unique single stranded cDNA will be amplified. Suppression subtractive hybridization (SSH) has recently been developed as a powerful tool to reduce the unwanted background (Diatchenko et al., Proc. Natl. Acad. Sci. USA 93 (1996), 6025-6030; Harms et al, Lett. Appl. Microbiol. 35 (2002), 113-116). First the cDNA is digested with a blunt cutting restriction enzyme, then the resulting tester cDNA fragments are subdivided into two samples (A and B) and ligated to the corresponding different adapters A and B at their 5 '-ends in separate tubes. During the first stage of subtraction, excess driver is added to each sample of tester and the samples are allowed to hybridize. After the subtraction hybridization, samples A and B (having different ligated adapters) are mixed and allowed to reassociate. This leads to the creation of novel molecules from the subtracted ssDNA that are by definition asymmetrically flanked by adapter A at one end and adapter B at the other. These molecules may now be isolated using primers that recognize the outer parts of adapters A and B. Amplification of symmetrically flanked tester cDNA fragments from the first stage will be suppressed, and hybrids formed with the driver will also not amplify due to the lack of one or both primer annealing sites, leading to preferential amplification of the asymmetrically flanked sequences of interest.

But even with this profiling technique the identification of differentially expressed genes that are present in the tester as well as the driver sample although with different abundance is very difficult. The background of non-differentially expressed genes reduces the number of false positive genes but due to the excess of driver DNA only genes are identified that are highly differentially expressed. The large number of genes that is truly differentially expressed but only to a moderate extent can not be identified. It remains a common problem of all methods for the comparison of expression profiles to identify genes within the pool of differentially expressed genes and markers that differ only slightly in their expression levels in test vs. control sample.

The present invention provides a method for enhancing the difference in expression of genes involved in a particular phenotypic trait and thereby aiding the identification of these genes by subtractive hybridization. Description of the invention

The present invention generally relates to a method for identifying and/or isolating a nucleic acid or a corresponding gene involved in or correlating with a phenotypic trait. Said method comprises effecting subtractive hybridization using a cDNA derived from transcript RNA of a test sample and an excess of a driver nucleic acid derived from a reference sample; wherein

(i) the cDNA of said test sample is obtained from subtractive hybridization using cDNA derived from transcript RNA of a first test sample that has been subjected to biotic or abiotic stress and an excess of RNA of a second non-treated test sample; and/or

(ii) the driver nucleic acids comprise RNA obtained from subtractive hybridization using RNA of a first reference sample that has been subjected to the same biotic or abiotic stress as said first test sample and an excess of cDNA derived from transcript RNA of a second non-treated reference sample; or (iii) the driver nucleic acids comprise a mixture of RNA obtained from said second non- treated test sample and RNA obtained from said first and second reference sample.

The method of the present invention is based on the triggering of RNA expression responsible for certain phenotypes by applying a specific stress or stimulus in combination with an improved subtractive hybridization procedure using chemical cross-linking of hybrids. It could be surprisingly shown, that the large number of candidate genes which appear to be differentially expressed using conventional methods, is reduced employing the method according to the invention. On the other hand, due to the challenging step genes of different expression capacity, which at first glance do not seem to be differentially expressed according to standard methods, can now be determined and isolated. The internal subtraction in combination with the triggering of the expression of the relevant genes leads to an enrichment of genes correlating with the tested phenotype. Thereby the effort for the subsequent characterization and verification of candidate genes is effectively reduced. The method of the present invention can be preferably performed according to two different approaches, depicted in Fig.l and Fig.2. Hybrid selection can be achieved in one step as shown in Fig.l, wherein the cDNA is derived from RNA from a test subject sample after stress application and RNA from an untreated subject sample as well as RNA from a subject not displaying the phenotypic trait with one sample treated like the test sample and one sample left untreated are used as drivers for subtractive hybridization.

The second approach involves two parallel hybrid selection steps, wherein the RNA from a test subject prior stress application is subtracted from the cDNA of said subject after stress application, leaving the cDNA molecules unique to the stress subjected test sample free to hybridize with RNA obtained by subtracting the cDNA of a control sample prior stress application from the RNA from said control after stress application.

Since a stabilization of DNA-RNA hybrids reduces the background by preventing already subtracted molecules to enter into the amplification process, the present invention further comprises effecting selective cross-linking of the nucleic acid strands of said cDNA and RNA in duplex molecules. Cross-linking of DNA.RNA duplexes can be achieved by different means. US-A-6,005,093, for example, discloses novel coumarin derivatives comprising a coumarin moiety linked to a non-nucleosidic backbone moiety. The resulting molecules can be used as photoactivate cross-linking groups when incorporated into polynucleotides as replacements for one or more of the complementary nucleoside bases. Incorporating said molecules into the cDNA during synthesis would provide such a selective cross-linking. US-A-5, 824,796 also describes novel substituted nucleotide bases with a crosslinking arm which accomplish crosslinking between specific sites on adjoining strands of oligonucleotides or oligodeoxynucleotides.

Non-substituted nucleic acid molecules can be cross-linked by employing chemical agents that interact with either the phosphate backbone or the bases of the double strand to form covalent links. US-A-5,591,575 discloses a chemical cross-linking agent useful for subtractive hybridization.

In a preferred embodiment of the methods of the present invention, the cross-linking agent is an aziridinylbenzoquinone, particularly 2,5-bis(l-aziridinyl)-3,6-bis(carbethoxyamino)-l,4- benzoquinone (AZQ), see e.g. Hampson et al, Nucleic Acids Res. 20 (1992), 2899. This bifunctional, alkylating chemical induces irreversible DNA:DNA and DNA:RNA interstrand bonds under participation of the N⁷ of guanidine residues. ^"Double strands formed by AZQ can not be separated by heat denaturation thereby effectively inhibiting their amplification in for instance a PCR. A general method for obtaining nucleic acid sequences using specific cross- linking is described in W099/18236.

In one embodiment of the method of the present invention said cross-linking is effected after said one or more selective hybridization steps. The test DNA or RNA can be hybridized repeatedly with the immobilized driver and thus insuring that all molecules represented in the driver hybridize to the respective complementary molecule in the test sample and are removed from the pool of single stranded molecules.

Repeated rounds of selective hybridization are best achieved if one of the hybridization partners is immobilized to further the separation of molecules still single stranded and those which have already hybridized. In one embodiment, the method of the present invention comprises immobilizing said RNA of said test sample and/or said cDNA of said second non- treated reference sample on a solid support.

A common procedure for immobilization of nucleic acid is simply binding it to a membrane. Nitrocellulose or nylon membranes are well known in the art as well as the methods for fixing the nucleic acids on them. For example, Amersham Biosciences distributes a variety of nylon and nitrocellulose membranes optimized for different hybridization and detection techniques with detailed protocols for nucleotide fixation. Nucleic acid molecules which have hybridized to the fixed nucleotides can be removed by conventional "stripping" procedures, described for instance in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press. The membrane is then used for a second round of hybridization with the test sample. Depending on the stringency of the "stripping", the membrane can be used several times, usually 3 to 10 hybridizations.

Another way for immobilizing nucleic acids involves binding them to a matrix like, for example sepharose, and immobilize the matrix in a chromatography column. By employing well known standard techniques, the test sample could be run several times over the column, regenerating the column between the runs and thereby providing repeated rounds of selective hybridization. Other matrices known in the art and employed to immobilize nucleic acids are glass or plastic surfaces, preferably in a chip or microarray format.

The single stranded DNA or RNA molecules obtained after selective hybridization will usually be in a very low concentration. Therefore in a preferred embodiment of the invention any one of the above described methods involves a subsequent amplification of the single stranded molecules obtained after subtractive hybridization, comprising

(a) adding to said mixture obtained after hybridization or cross-linking an effective amount of reagents necessary for performing an amplification reaction; and

(b) cycling the mixture obtained after step (a) through at least one cycle of the denaturing, annealing and primer extension steps, wherein amplification of hybridized and/or cross- linked nucleic acid molecules is suppressed during the amplification reaction. While cross-linking of hybrids suppresses the amplification of these molecules by preventing the denaturation of double strands and thereby not supplying a template single strand for the annealing and primer extension steps, non-cross-linked hybrids will serve as a template and are removed prior to amplification. Several techniques are known in the art to remove hybridized molecules from a mixture, including immobilizing one of the hybridization partners as described above, employing streptavidin-coated beads to remove biotin-labeled molecules or selective enzymatic digestion of double strands, techniques which are well known to those skilled in the art. Although random priming oligonucleotides can be used to amplify single stranded molecules after hybridization or cross-linking, a preferred embodiment of the method of the invention further comprises attaching PCR adapters to each end of remaining non-hybridized cDNAs after selective hybridization.

The use of defined PCR adapters has several advantages. Adapters provide 5 '- and 3 '-ends with known sequence to the molecules which facilitates subsequent manipulation. Adapters can be designed to contain restriction endonuclease recognition sites enabling cloning of the remaining non-hybridized cDNAs and provide sites for sequencing primers or primers for further rounds of amplification. Such primers will often be nested primers, provided that the adapters are long enough. The adapters can be attached to the ends of DNA or RNA fragments using a variety of techniques that are well known in the art, including DNA ligase- mediated ligation of the adapters to sticky- or blunt-ended DNA, T4 RNA ligase-mediated ligation of a single-stranded adapter to single-stranded RNA or DNA, oligo (dA) tailing using terminal transferase, or via any DNA polymerase (or a reverse transcriptase if RNA is the template) using a primer having a sequence which corresponds to the adapter sequence. As used herein, the term "attach," when used in the context of attaching the adapter to a DNA fragment, refers to bringing the adapter into covalent association with the DNA fragment regardless of the manner or method by which the association is achieved. Preferably, the adapter should not contain any sequences that can result in the formation of "hairpins" or other secondary structures in the DNA which can prevent adapter ligation or primer extension. As would be readily apparent to a person skilled in the art, the primer binding sequence portion of the adapter can be complementary with a PCR primer capable of priming for PCR amplification of a target DNA.

Preferably, the primers of the subject invention have exact complementarity with the adapter sequence. However, primers used in the subject invention can have less than exact complementarity with the primer binding sequence of the adapter as long as the primer can hybridize sufficiently with the adapter sequence so as to be extendable by a DNA polymerase. As used herein, the term "primer" has the conventional meaning associated with it in standard PCR procedures, i.e., an oligonucleotide that can hybridize to a polynucleotide template and act as a point of initiation for the synthesis of a primer extension product that is complementary to the template strand.

Design of adapters and primers as well as the choice of appropriate hybridization conditions can be performed according to known methods , see, e.g., Nucleic Acid Hybridization (1985) Ed. James, B. D. & Higins, S. J. (IRL Press Ltd., Oxford) and the appended examples.

Using the teachings contained herein and in the prior art, the person skilled in the art could readily construct adapters useful for the method according to the invention. The adapters and primers used in the subject invention can be readily prepared by the person skilled in the art using a variety of techniques and procedures. For example, adapters and primers can be synthesized using a DNA or RNA synthesizer. In addition, adapters and primers may be obtained from a biological source, such as through a restriction enzyme digestion of isolated DNA. The primers can be either single- or double-stranded. Preferably, the primers are single stranded. In a particular preferred embodiment of the methods of the present invention, said adapters or nucleic acid primers comprise a nucleotide sequence comprising a restriction endonuclease recognition site.

Since cross-linking effectively inhibits the amplification of hybridized molecules, it is possible to attach said adapters before or after cross-linking of said cDNA and RNA.

In a further embodiment the method of the present invention comprises a step of second strand synthesis. The cDNAs remaining after hybridization can be subjected to synthesis of the complementary strand either by employing one of more cycles of a usual PCR, as described above, or by other means of primer extension.

In the challenging step, said biotic stress can be, for example, a pathogen infection or a disease. Test and control subject can react differently to infection with the same pathogen or upon suffering from the same disease depending on the phenotypic traits of the subjects. Depending on their respective genetic characteristics test and control subject can activate different mechanisms to fight the infection or disease. For example cells of the human immune system from different individuals might be infected with HIV in vitro to identify genes that contribute to some individuals remaining symptomless for more than 15 years although infected with HIN, while others succumb to AIDS within a few years of becoming infected. Animal models such as mice, rats, dogs, can be used to induce a certain disease such as a vascular disease and subsequently analyzed for heart protecting genes. Another example would be the infection of plants with a plant virus, as is well known by those skilled in the art e.g. either by aphid-mediated infection or direct application onto the leaves. Other biotic stresses include parasite infection or mould and other fungal infections or mycoses.

In embodiments of the present invention, wherein abiotic stress is used as a challenge said stress can be for example, environmental stress, salt stress, drought, starvation, drug exposure, or a noxious challenge.

If not stated otherwise the terms "drug", and "noxious challenge" include but are not limited to therapeutic agents (or potential therapeutic agents), agents of known toxicities such as neurotoxins, hepatic toxins, toxins of hematopoietic cells, myotoxins, carcinogens, teratogens, or toxins to one or more reproductive organs. The noxious challenge can further be agricultural chemicals, such as pesticides, fungicides, nematicides, and fertilizers, cosmetics, including so-called "cosmeceuticals", industrial wastes or by-products, or environmental contaminants. They can also be animal therapeutics or potential animal therapeutics. Household products that can be used in accordance with the methods of the present invention include bleaches, toilet, blocks, washing-up liquids, soap powders and liquids, fabric conditioners, window, oven, floor, bathroom, kitchen and carpet cleaners, dishwater detergents and rinse aids, watersoftening agents, descalers, stain removers, polishes, paints, paint removers, glues, solvents, varnishes, air fresheners, moth balls and insecticides. New ingredients for household products are constantly being developed and can be employed as challenge. For example, in recent years new enzymes (to digest stains) and "optical brighteners" (which make washing appear whiter) have been developed for use in washing powders and liquids. New surfactants (which cut through grease to remove ingrained dirt) and chemical "builders" (which act as water softeners and enable surfactants to work more effectively) have been developed for use in washing powders and liquids, washing-up liquids and various cleaning agents. But also medical materials can be used, for example dental materials such as new filling polymers, metal alloys, and bioactive ceramics. Furthermore, chemical compositions of any part of a device, such as the electrode and/or electrode, adhesives, paste, gel or cream including the concentrations of the different ingredients and impurities present may be used as a challenge in the method of the present invention. Again, test and control subject might react differently depending on their respective genetic background to all sorts of abiotic stress. It is for instance well known phenomenon that humans react quite differently to the same drug. While in some patients a drug might be beneficial, it can cause severe side effects in others while being of little or no therapeutic use. Other examples include bacteria strains with different tolerance for increased temperature or the absence of certain nutrients. Those skilled in the art will readily appreciate that the applied stress has to be appropriate to trigger the expression of genes responsible for the phenotype to be tested.

In a particularly preferred embodiment of the method of the invention, said noxious challenge is radiation either by UV-light or X-rays. Both types of radiation are known to cause different kinds of DNA damage which induces a variety of repair mechanisms. Organisms of the same species often show individually different responses to such a noxious challenge, reflecting the individual ability to repair DNA damage and thereby giving an indication of the overall resistance to DNA damage caused disorders like cancer.

In another preferred embodiment of the present invention, the described method is performed with a tester and/or driver nucleic acid sample comprising a pool of nucleic acids. This measure is particularly useful for the identification of genes which are most likely responsible for a certain phenotype. For example, in order to identify a disease causing gene a tester nucleic acid sample obtained from a patient is screened against a driver nucleic acid sample comprising nucleic acids from several healthy subjects of different cultural background in order to exclude the amplification of nucleic acid sequences that are unique simply because of lineage and descent of an individual. In a similar manner the tester nucleic acid sample might be obtained from a pooled DNA sample of population of subjects, while the driver nucleic acid sample is derived from an individual subject in order to identify genes that vary within said population. By applying pooled nucleic acid samples for the tester as well as for the driver to the methods according to the invention the genetic characteristics of two populations can be compared.

The nucleic acid tester and driver samples can be derived from cells, tissue or organisms displaying different phenotypes such as a symptom of a disease. However, it is to be understood that the methods of the present invention are also particularly useful for the identification and isolation of "hidden" nucleic acids, which do not or at least not at the onset of their presence display an observable phenotype, for example in genetic predispositions, contamination of foods, and infected animals. It can also be employed for the selection of breeding animals or seeds for agricultural crops to select specific desired traits without employing transfer of genetic material from other species. The methods of the present invention are particularly powerful when samples are used, which are derived from the same or similar species, in particular if said samples are derived from the same or closely related subjects, for example twins.

Since the method of the present invention has been proven to be particularly useful for the analysis of complex genomes, the test samples are preferably derived from a vertebrate or a plant. In the latter embodiment, the methods of the present invention are especially useful in plant breeding, for example in identifying pathogen resistance genes. In the preceding embodiment, said vertebrate is preferably a mammal or a fish; particularly human is preferred. In the field of pharmacogenomics the methods of the invention allow the identification of molecular markers that e.g. confer the development of undesired side effects of a certain drug in an individual or determine the efficacy of a medicament in an individual.

In one important aspect of the present invention, the methods described herein are employed to identify genes that are etiologically related to a disease. With this technique, DNA samples from disease specimens will be hybridized with samples from normal specimens to identify DNA sequences that are present or absent the disease specimens. These sequences will be analyzed further to elucidate their functions that may be causally related to the disease. Accordingly, in this embodiment said tester nucleic acid sample is derived from diseased tissue and said driver nucleic acid sample is derived from healthy tissue or vice versa. Hence, it is expected that said nucleic acid molecules identified by a method of the present invention to be unique to the tester sample correspond to disease causing genes.

In a preferred embodiment of the present invention, said unique nucleic acid or corresponding gene identified or isolated is present in the diseased tissue and absent in the healthy tissue or vice versa. Furthermore, information generated from the methods according to the invention can be used to design DNA arrays and/or chips which allow monitoring populations e.g. for their clinical role of the genes for the same disease in different regions around the world, early diagnosis of disease, response to therapy, or assessment of health risk. Chip and array technology are well known to the person skilled in the art. Advances in approaches to DNA- based diagnostics are reviewed, for example, by Whitcombe et al. in Curr. Opin. Biotechnol. 9 (1998), 602-608. Furthermore, DNA chips and microarray technology devices, systems, and applications are described by, e.g. Cuzin, Transfus. Clin. Biol. 8 (2001), 291-296 and Heller, Annu. Rev. Biomed. Eng. (2002), 129-153. Furthermore, active microelectronic array systems for DNA hybridization, genotyping and pharmacogenomic applications (see, e.g., Sosnowski, Psychiatr. Genet. 12 (2002), 181-192) can be employed in accordance with the present invention.

However, as mentioned before, the methods of the present invention are not restricted to analysis of disease related phenotypes but encompass the analysis of any genotypic difference between at least two samples. Those subjects may differ also in their phenotype which may be any phenotype that can be recognized or measured in any way. Those phenotypes typically include economically important phenotypes, i.e. traits, in particular if those traits are multigenetically inherited. This makes the method of the present invention also useful in plant and animal breeding.

The methods of the present invention can further comprise the step of cloning and/or sequencing the identified nucleic acid fragments. Detailed descriptions of conventional methods, such as those employed in sequencing, the construction of vectors and plasmids, the insertion of genes encoding polypeptides or the corresponding antisense constructs into such vectors and plasmids, the introduction of plasmids into host cells, and the expression and determination thereof of genes and gene products can be obtained from numerous publications, including Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press. Candidate nucleic acids or encoded polypeptides identified in such a manner can be validated by expressing them and observing the phenotype.

Overexpression or inhibition of expression of the identified candidate nucleic acid or encoded polypeptide in said cell, tissue or animal indicates whether the candidate is capable of inducing a responsive change in the phenotype which is preferably related to a disorder. The responsive change in the phenotype of said cells can be observed by subjecting the cells, secreted factors thereof, or cell lysates thereof, to analyze different parameters like cell proliferation, electrophysiological activity, DNA synthesis, out-growth of cells, cell migration, chemokinesis, chemotaxis, development of vessels, marker gene expression or activity, apoptosis and/or vitality, etc. Hence, said identified, sequenced and/or cloned nucleic acid fragment preferably belongs to an infectious agent, a food contaminant, a gene responsive to the presence, sensitivity or resistance to toxicants, health risk, or a gene involved in a disease. Most preferably, said disease is cancer, hypertension, or diabetes.

In case the nucleic acid sequence of the amplified DNA fragment does relate to an unknown gene, it is envisaged to clone the corresponding gene and to elucidate its function. Thus, in a further embodiment the method of the present invention further comprises using the identified, sequenced and/or cloned nucleic acid fragment as a probe for cloning the corresponding gene or full length cDNA. Methods which are well known to those skilled in the art can be used to obtain and screen genomic or cDNA libraries; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Furthermore, various DNA libraries are commercially available; see, e.g., Clontech.

In another embodiment the present invention relates to a method for diagnosing in a subject a phenotype, preferably disease or a predisposition to such a phenotype comprising:

(a) analyzing a sample of nucleic acids of a subject for example by means of a diagnostic chip, primer extension, single nucleotide polymorphisms, probe or sequencing comprising a nucleic acid molecule identified or cloned as described above, and

(b) comparing the result with that of a sample obtained from a subject displaying or known to develop the phenotype, wherein the presence or absence of said nucleic acid or the corresponding gene or cDNA is indicative for the phenotype or a corresponding predisposition. Similarly, a corresponding method may be used for analyzing a sample for the expression product of the mentioned nucleic acid molecule, for example by means of antibody.

In these embodiments, nucleic acid molecules, (poly)peptides, or antibodies are preferably detectably labeled. A variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention. Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immuno assays", Burden, RH and von Knippenburg (Eds), Volume 15 (1985), "Basic methods in molecular biology"; Davis LG, Dibmer MD; Battey Elsevier (1990), Mayer et al, (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London(1987), or in the series "Methods in Enzymology", Academic Press, Inc. There are many different labels and methods of labeling known to those of ordinary skill in the art. Commonly used labels comprise, inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes (like P or I), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums). Labeling procedures, like covalent coupling of enzymes or biotinyl groups, iodinations, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases) are well known in the art. Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.

In addition, the above-described nucleic acids, proteins, antibodies, etc. may be attached to a solid phase. Solid phases are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, animal red blood cells, or red blood cell ghosts, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes. Suitable methods of immobilizing nucleic acids, (poly)peptides, proteins, antibodies, etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions and the like. The solid phase can retain one or more additional receptor(s) which has/have the ability to attract and immobilize the region as defined above. This receptor can comprise a charged substance that is oppositely charged with respect to the reagent itself or to a charged substance conjugated to the capture reagent or the receptor can be any specific binding partner which is immobilized upon (attached to) the solid phase and which is able to immobilize the reagent as defined above.

Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. These comprise, inter alia, RIA (Radioisotopic Assay) and IRMA (Immune Radioimmunometric Assay), EIA (Enzym Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno Assay), CLIA (Chemioluminescent Immune Assay), and electronic chip and array systems; see supra. Other detection methods that are used in the art are those that do not utilize tracer molecules'. One prototype of these methods is the agglutination assay, based on the property of a given molecule to bridge at least two particles. For diagnosis and quantification of (poly)peptides, polynucleotides, etc. in clinical and/or scientific specimens, a variety of immunological methods, as described above as well as molecular biological methods, like nucleic acid hybridization assays, PCR assays or DNA Enzyme Immunoassays (Mantero et al., Clinical Chemistry 37 (1991), 422-429) have been developed and are well known in the art. In this context, it should be noted that the nucleic acid molecules may also comprise PNAs, modified DNA analogs containing amide backbone linkages. Such PNAs are useful, inter alia, as probes for DNA/RNA hybridization. Further diagnostic methods leading to the detection of nucleic acid molecules in a sample comprise, e.g., polymerase chain reaction (PCR), ligase chain reaction (LCR), Southern blotting in combination with nucleic acid hybridization, comparative genome hybridization (CGH) or representative difference analysis (RDA). These methods for assaying for the presence of nucleic acid molecules are known in the art and can be carried out without any undue experimentation.

The subject invention further concerns kits and compositions which contain, typically in separate packaging or compartments, the reagents such as driver nucleic acid samples, adapters and primers required for practicing the PCR suppression method of the subject invention. Such kits may optionally include the reagents required for performing PCR reactions, such as DNA polymerase, DNA polymerase cofactors, and deoxyribonucleotide-5'- triphosphates. Optionally, the kit may also include various polynucleotide molecules, DNA or RNA ligases, restriction endonucleases, reverse transcriptases, terminal transferases, various buffers and reagents, and antibodies that inhibit DNA polymerase activity. The kits may also include reagents necessary for performing positive and negative control reactions. The kit may also contain components for high throughput screening (HTS) such as microarrays, chips, multi-well plates and apparatuses therefor. Optimal amounts of reagents to be used in a given reaction can be readily determined by the skilled artisan having the benefit of the current disclosure.

The above described methods and kits can be used for e.g. in monitoring food or seed, forensic analysis, pharmacogenomics, tumor diagnostics, marker assisted breeding, quality control of raw material and seed or analysis of differences of closely related organisms. Moreover, the methods of the present invention can be used for improving drug response with pharmacogenomics. Adverse drug reactions, which in the USA are estimated to account for 100,000 hospitalizations annually, could be halved by the implementation of personalized medicine, for example by analyzing a patient with a method of the present invention for the presence or absence of expression of a gene involved in drug metabolism; see for review, e.g., Ferentz, Pharmacogenomics 3 (2002), 453-467. Thus, the method of the present invention can be applied advantageously throughout drug development to bring drugs successfully to market along with diagnostic tests that ensure their appropriate use.

These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the materials, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices. For example the public database "Medline" may be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples and figure which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature; see, for example, DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridisation (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986). Detailed descriptions of conventional methods, such as those employed in the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids, the introduction of plasmids into host cells, and the expression and determination thereof of genes and gene products can be obtained from numerous publications, including Sambrook et al, (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press.

The figures show:

Figure 1: One-step subtractive hybridization Hybrid selection can be achieved in one step. Tester and driver samples with different phenotypic trait(s) are divided into two halves and subjected to an abiotic or biotic stress while the other half remains untreated or treated differently. After RNA isolation the RNA from the tester sample after stress application is transcribed into cDNA and RNA from the tester sample prior stress application as well as RNA from the two driver samples displaying a different phenotypic trait are used for subtractive hybridization.

Figure 2: Two-step subtractive hybridization This approach involves two parallel hybrid selection steps, wherein the RNA from an untreated tester sample is subtracted from the cDNA of said sample after stress application, leaving the cDNA molecules unique to the stress subjected tester sample free to hybridize with RNA obtained by subtracting the cDNA of an untreated driver sample from the RNA from said driver after stress application. Figure 3: Identification of genes responsible for resistance to lung cancer Whole blood from a population of unrelated healthy smokers is pooled, divided into two samples and challenged by X-ray exposure or left untreated. Whole blood from a smoking lung cancer patient too is divided into two samples and challenged by radiation, receiving the same dose. After a recovery phase to allow induction of repair processes lymphocytes are isolated by standard methods, their RNA is isolated and the RNA from the irradiated smoking control group transcribed into cDNA and used as a tester DNA while the RNA from the other three samples is used as driver in the hybrid selection. After chemical cross-linking, adapters are ligated, the cDNA remaining single stranded is subjected to second strand synthesis and amplified by PCR. Amplified genes and gene fragments are identified by sequencing.

Figure 4: Identification of genes responsible for resistance to skin cancer Skin fibroblasts from a skin cancer patient and his healthy sibling are divided into two samples each and challenged by radiation with UV light and left untreated, respectively. After a recovery phase to allow induction of repair processes the fibroblasts are washed 3 times in ice-cold phosphate buffered saline (PBS), harvested by scraping and pelleted. RNA is isolated from all four samples and two parallel hybrid selections are performed. In case of the samples from the healthy sibling which served as tester sample the RNA from the non-irradiated half is immobilized on a nylon membrane while the irradiated half is subjected to a reverse transcription. The hybrid selection leaves the cDNA unique to the irradiated tester sample free to hybridize with RNA unique to the irradiated driver sample from the skin cancer patient. Said unique RNA is obtained by immobilizing the cDNA transcribed from the RNA of the non-irradiated driver sample and hybridizing the membrane with the RNA from the irradiated driver sample. The unique RNA thus obtained is hybridized with the cDNA from the first tester hybridization, ligated to adapters, and following chemical cross-linking subjected to second strand cDNA synthesis. Only cDNA unique to the irradiated sample from the healthy person are able to take part in the synthesis and be further amplified by PCR. Amplified genes and gene fragments are identified by sequencing. EXAMPLES

Example 1: Characterization of genes differing between a lung cancer patient and a control group of smokers after challenge with x-rays

Aim of this study was the identification of genes protecting a smoker from developing lung cancer. While direct comparison of two gene pools e.g. by differential display or RNA footprinting (Konietzko and Kuhl, Nucl. Acid Res. 26 (1998), 1359-1361; Saito et al, Mol. Pathol. 55 (2002), 34-39) leads to a large number of candidate genes which are differentially expressed, the method according to the invention employs an internal subtraction to reduce the number of genes not correlating with the tested phenotype. Thereby the effort for the subsequent characterization and verification of candidate genes is effectively reduced.

1.1 Induction of the repair capacity Whole blood from 20 unrelated healthy smokers was pooled, divided into two samples and challenged by radiation with 1,66 Gy (Gray) or left untreated. Whole blood from a smoking lung cancer patient too was divided into two samples and challenged by radiation, receiving the same dose (Fig.3). After a recovery phase of from 5-30 min at 37°C to allow induction of repair processes lymphocytes were isolated by standard methods (Boyum, Scand J Immunol., Suppl 5 (1976), 9-15).

1.2 RNA-Isolation

Total RNA from the irradiated control group lymphocytes was isolated by the Guanidinium thiocyanate method (Chomczynski and Sacchi, Anal. Biochem. 162 (1987), 156; Chomczynski, BioTechniques 15 (1993), 532-537; Chomczynski and Mackey, BioTechniques 19 (1995), 924-945) followed by isolation using an oligo-dT cellulose column (Sambrook et al, (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press). In a similar manner Poly(A)+ mRNA was isolated from the remaining samples.

1.3 first Strand cDNA-Svnthesis

Poly(A)+ mRNA from irradiated control lymphocytes was subjected to reverse transcription to obtain single stranded cDNA using the Promega kit (ImProm-II™ Reverse Transcription System) according to the manufacturers instructions. 1.4. Expression capacity- Analysis

In order to monitor the effect of radiation on the expression pattern of lymphocytes and to optimize the dose to induce repair mechanisms the expression of two genes known to become upregulated after exposure to X-rays was determined.

The XRCC (X-ray repair cross complementing group) comprises a number of genes that are induced after irradiation and take part in the repair of DNA damages. The following conditions were used:

l x PCR buffer:

50 mM KC1, lO mM Tris-HCl,

1.5 mM MgCl₂ ,

0.1 % Triton X- 100,

All reactions were performed in a total volume of 50μl containing:

50 ng 1 Strand cDNA (appr. 5μl)

20 μM dNTPs,

0,5 pmol of each primer 0,25 units Taq polymerase

-Amplification was carried out as followed: 5 min initial denaturation at 95°C followed by 35 cycles: 95°C for 45 sec denaturation 45 sec annealing temperature of primer (see below) 68°C 1 min elongation final extension at 68°C for 7 min.

Annealing-Temperatures of the repair gene primers: l. XRCC1 55°C 2. XRCC3 60°C

Only samples that received a dose sufficient to induce XRCC 1 and 3 were subjected to hybridization. 1.4 Liquid-Liquid-Hybridisation

3-5 μg cDNA and 10 μg Poly(A)⁺ RNA from each of the three remaining samples (non- irradiated control lymphocytes; patient lymphocytes with or without radiation challenge) were denatured and mixed in a volume of 1 Oμl hybridization buffer. Hybridization was carried out over night at 68°C.

Hyb.-buffer: 0, 5 M NaCl 25 mM Hepes, pH 7,5 5 mM EDTA

Resuspension buffer 25 mM Tris-HCl, pH 7,5 I mM EDTA 5 % DMSO 2 mM Ascorbic acid 1 % SDS

After hybrization the RNA/cDNA mixture was diluted in 5 volumes of sterile water (DNA grade) and precipitated with three time vol. of ethanol.

1.5 Chemical cross-linking

To eliminate the hairpin structures of the single stranded cDNA the precipitated RNA-cDNA- complex was dissoved in 50μl resuspension buffer and incubated at 68°C for three min. Subsequently, the incubation temperature was reduced^' to 45°C and 2,5 diaziridinyl-1,4- benzoquinone in a concentration of 200μM was added and the mixture was incubated for 20 min to allow cross-linking.

1.6 Results

To verify that the combination of challenge and subtractive hybridization is applicable to identify inherited differences between control group and patients polymerase chain reaction (PCR) followed by enzymatic digestion was used for genotyping of the XRCCl-Arg399Gln, XRCC3-Thr241Met and XPD-Lys751 Gin mutations according to (Shen, et al, Cancer Res. 58, (1998), 604-608). All primers used for genotyping were synthesized by Sigma and were composed of sense, 5 '-CAAGTACAGCCAGGTCCTAG-3 ' (SEQ ID NO: 1); antisense, 5'-CCTTCCCTCATCTGGA-GTAC-3 ' (SEQ ID NO: 2) for XRCC1; sense 5'-GCCTGGTGGTCATCG-ACTC-3 ' (SEQ ID NO: 3); antisense, S'-ACAGG'-GCTCTGGAGGCACTGCTCAGCTCACGCACC-S' (SEQ ID NO:

4); for XRCC3 and sense, S^{^}-CTGCTC-AGCCTGGAGCAGCTAGAATCAGAGGAGAG-GAGACGCTC-S ' (SEQ ID NO: 5) antisense S'-AAGACC-TTCTAGCACCACCG-S ' (SEQ ID NO: 6) for XPD, respectively.

The 50μl PCR reaction volume contained 1 x PCR buffer (50mM KC1, lOmM Tris-HCl, l,5mM MgCl₂ , 0,1% Triton X-100, 20μM dNTPs, 0,5 pmol of each primer, 0,25 units of Platinum Proof Reading Taq polymerase (Gibco, Life Science) and 50ng of DNA. Using a MJ

Research MultiCycler PTC 220 Dyad thermal cycler, the PCR mixture was amplified using a

5min denaturation step at 95°C followed by 35 cycles consisting of 95°C for 45 s, 45 s at the appropriate annealing temperature and 68°C for 1 min followed by a final extension step at

68°C for 7 min. The XRCCl-Arg 399Gln polymorphism, a G to A transitions in exon 10, the XPD-Lys751 Gin polymorphism, an A to C transversion in exon 23 and the XRCC3-Thr Met polymorphism, a T to C transition was determined as described at (Matullo et al.

Carcinogenesis 9 (2001), 1437-1445). The 248 bp XRCC1-PCR product was digested with

Neil: the allele was cut into 89 and 159 bp fragments (Gin allele not digested). The XPD-PCR product was digested with Pstl: the Lys allele was cut into 41 and 120 bp fragments (Gin allele not digested) and the XRCC3-PCR product with was digested with Ncol: The Thr allele was cut into 39 and 97 bp fragments (Met allele not digested). The restriction enzymes were provided by Promega.

The XRCC3-Thr Met polymorphism was identified in the lung cancer patient thus proving the validity of the approach. To identify unknown genes and mutations free cDNA after chemical crosslinking is ligated to adapters, amplified by PCR using primers annealing to the adapters and the PCR products are sequenced by conventional cycle sequencing; see figure 3. Example 2: Characterization of minute differences between fibroblasts from a skin cancer patient and his sibling after challenge with UV-light

This experiment was performed to verify the method of the invention with another abiotic stress applied to different cells.

2.1 Induction of the repair capacity

Skin firoblasts from skin cancer patient and his healthy sibling were divided into two samples each and challenged by radiation with UV light (302 nm) and left untreated, respectively. (Fig.4). After a recovery phase of 15 min at 37°C to allow induction of repair processes the fibroblast were washed 3 times in ice-cold phosphate buffered saline (PBS), harvested by scraping and pelleted.

2.2 RNA-Isolation Total RNA from all four skin fibroblast pellets was isolated by the Guanidinium thiocyanate method followed by isolation using an oligo-dT cellulose column as described in Example 1.

2.3 first Strand cDNA-Synthesis

Poly(A)+ mRNA from irradiated control fibroblasts was subjected to reverse transcription to obtain single stranded cDNA as described in Example 1.

2.4. Expressions capacity-Analysis

In order to monitor the effect of UV-light exposure on the expression pattern of skin fibroblasts and to optimize the dose to induce repair mechanisms the expression of a gene known to become upregulated after exposure to UV radiation was determined.

The XPD (xeroderma pigmentosum complementary group D) system includes a number of genes that are induced specifically after irradiation with light of short wave length.

PCR was performed as in Example 1 except for the annealing temperature of the XPD gene primers which was 67°C. Only samples that received a dose sufficient to induce XPD were subjected to hybridization. 2.5 Liquid-Liquid-Hybridisation

3-5 μg cDNA and 10 μg Poly(A)+ RNA from each of the three remaining samples (non- irradiated control lymphocytes; patient lymphocytes with or without radiation challenge) were denatured and mixed in a volume of lOμl hybridization buffer. Hybridization and subsequent precipitation was carried out as in Example 1.

2.6 Chemical cross-linking

To eliminate the hairpin structures of the single stranded cDNA the precipitated RNA-cDNA- complex was dissoved in 50μl resuspension buffer and incubated at 68°C for three min. Subsequently, the incubation temperature was reduced to 45°C and 2,5 diaziridinyl-1,4- benzoquinone in a concentration of 200μM was added and the mixture was incubated for 20 min to allow cross-linking.

1.6 Results PCR was performed as in example 1 and an XPD polymorphism was identified in the patient sample. Subsequent steps to identify further unknown genes involved in the genesis of skin cancer include the ligation to adapters, amplification by PCR using primers annealing to the adapters and the sequencing of the PCR products.

Summary

The present invention works by triggering the increased expression of genes that cause a phenotypical difference and employs a modified subtractive hybridization assay to subsequently identify and isolate said genes or gene fragments. The presented methods are particular useful for the identification of novel genes involved in the development or prevention of various diseases, including cancer, hypertension and diabetes as well as for monitoring animals and food, for example for desirable breeding traits, infection and contaminants .

Claims

Claims

1. A method for identifying and/or isolating a nucleic acid or a corresponding gene involved in or correlating with a phenotypic trait, comprising effecting subtractive hybridization using cDNA derived from transcript RNA of a test sample and an excess of a driver nucleic acid derived from a reference sample; wherein (i) the cDNA of said test sample is obtained from subtractive hybridization using cDNA derived from transcript RNA of a first test sample that has been subjected to biotic or abiotic stress and an excess of RNA of a second non- treated test sample; and/or (ii) the driver nucleic acids comprise RNA obtained from subtractive hybridization using RNA of a first reference sample that has been subjected to the same biotic or abiotic stress as said first test sample and an excess of cDNA derived from transcript RNA of a second non-treated reference sample; or (iii) the driver nucleic acids comprise a mixture of RNA obtained from said second non-treated test sample and RNA obtained from said first and second reference sample.

2. The method of claim 1, -further comprising effecting selective cross-linking of the nucleic acid strands of said cDNA and RNA in duplex molecules.

3. The method of claim 2, wherein said cross-linking is effected after said one or more selective hybridization steps.

4. The method of claim 2 or 3, wherein the cross-linking agent is a aziridinylbenzoquinone .

5. The method of claim 4, wherein the cross-linking agent is 2,5-bis(l-aziridinyl)-3,6- bis(carbethoxyamino)- 1 ,4-benzoquinone (AZQ).

6. The method of any one of claims 1 to 5, wherein said RNA of said test sample and/or said cDNA of said second non-treated reference sample is immobilized on a solid support.

7. The method of any one of claims 1 to 6, further comprising (a) adding to said mixture obtained after hybridization or cross-linking an effective amount of reagents necessary for performing an amplification reaction; and (b) cycling the mixture obtained after step (a) through at least one cycle of the denaturing, annealing and primer extension steps, wherein amplification of hybridized and/or cross-linked nucleic acid molecules is suppressed during the amplification reaction;

8. The method of any one of claims 1 to 7 further comprising after selective hybridization attaching PCR adapters to each end of remaining non-hybridized cDNAs.

9. The method claim 8, wherein said adapters are attached before or after cross-linking of said cDNA and RNA.

10. The method of any one of claims 7 to 9 further comprising a step of second strand synthesis.

11. The method of any one of claims 1 to 10, wherein said biotic stress is pathogen infection, and/or disease.

12. The method of any one of claims 1 to 10, wherein said abiotic stress is environmental stress, salt stress, drought, starvation, drug exposure, and/or a noxious challenge.

13. The method of claim 12, wherein said noxious challenge is radiation by UV-light or X-rays.

14. The method of any one of claims 1 to 13, wherein the driver and/or tester nucleic acid sample comprises a pool of nucleic acids.

15. The method of any one of claims 1 to 14, wherein said samples are derived from cells, tissue or organisms which display different phenotypes.

16. The method of any one of claims 1 to 15, wherein said samples are derived from the same or similar species.

17. The method of any one of claims 1 to 15, wherein said samples are derived from the same subject or closely related subjects.

18. The method of any one of claims 1 to 17, wherein said samples are derived from a vertebrate or a plant.

19. The method of claim 18, wherein said vertebrate is a mammal or a fish.

20. The method of claim 19, wherein said mammal is a human.

21. The method of any one of claims 1 to 20, wherein said test sample is derived from diseased tissue and said driver nucleic acid sample is derived from healthy tissue or vice versa. _,

22. The method of any one of claims 1 to 21, wherein said nucleic acid corresponds to a disease causing gene.

23. The method of claim 21 or 22, wherein said nucleic acid or corresponding gene is present in the diseased tissue and absent in the healthy tissue or vice versa.

24. The method of any one of claims 7 to 23, wherein said adapters or nucleic acid primers comprise a nucleotide sequence comprising restriction endonuclease recognition site.

25. The method of any one of claims 1 to 24, further comprising cloning and/or sequencing the identified nucleic acid.

26. The method of any one of claims 1 to 25, wherein said identified, sequenced and/or cloned nucleic acid belongs to an infectious agent, a food contaminant, a gene responsive to the presence, sensitivity or resistance to toxicants or drugs, health risk, or a gene involved in a disease.

27. The method of any one of claims 1 to 26 further comprising using the identified, sequenced and/or cloned nucleic acid as a probe for cloning the corresponding gene.

28. A method for diagnosing in a subject a phenotype, preferably a disease or a predisposition to such a phenotype comprising: (a) analyzing a sample of a subject for the presence or absence of the nucleic acid or the corresponding gene identified and/or cloned by the method of any one of claims 1 to 27 or for the encoded gene product; optionally (b) comparing the result with that of a sample obtained from a subject displaying or known to develop the phenotype; wherein the presence or absence of said nucleic acid, the corresponding gene or gene product is indicative for the phenotype or a corresponding predisposition.

29. A kit for use in a method of any one of claims 1 to 28 comprising one or more components selected from the group consisting of a driver nucleic acid sample, restriction endonucleases, adapters, polymerases, primers, PCR reagents, microarray, chip, multi-well plate, a nucleic acid primer or adapter, a nucleic acid or gene obtained by the method of any one of claims 1 to 27.

30. Use of a method of any one of claims 1 to 27 or a kit of claim 29 for monitoring food or seed, forensic analysis, pharmacogenomics, tumor diagnostics, marker assisted breeding, quality control of raw material and seed or analysis of differences of closely related organisms.