MXPA01004550A - METHOD FOR INDEXING AND DETERMINING THE RELATIVE CONCENTRATION OF EXPRESSED MESSENGER RNAs. - Google Patents

Abstract

An improved method for the simultaneous sequence-specific identification of mRNAs in a mRNA population allows the visualization of nearly every mRNA expressed by a tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA. In general, the method comprises the formation of cDNA using anchor primers to fix a 3'-endpoint, producing cloned inserts from the cDNA in a vector containing a bacteriophage-specific promoter for subsequent RNA synthesis, generating linearized fragments of the cloned inserts, preparing cRNA, transcribing cDNA from the cRNA and performing two sequence specific PCR amplifications of the cDNA. In preferred embodiments, the method comprises comparing the length and at least part of the nucleotide sequence of the PCR products to expected values determined from a database of nucleotide sequences. The method can identify changes in expression of mRNA associated with the administration of drugs or with physiological or pathological conditions. Also provided are vectors and primers useful for the practice of the improved method.

Description

METHOD FOR CLASSIFYING AND DETERMINING THE RELATIVE CONCENTRATION OF EXPRESSED MESSAGING ARNES BACKGROUND OF THE INVENTION This invention is directed to methods for simultaneous identification of differentially expressed mRNAs, as well as measurements of their relative concentrations. A complete characterization of the protein molecules that make up an organism could be useful, for example for an improved drug design, the selection of an optimal treatment of individual patients and for the development of the most compatible biomaterial. Such characterization of expressed proteins can include their identification, determination of sequence, demonstration of their anatomical sites of expression, elucidation of their biochemical activities and understanding of how these activities determine the physiology of the organism. For medical applications, the description may also include information about how the concentration of each protein changes in response to pharmaceutical or toxic agents. Consider first the scope of the problem: How many genes are there? The issue of how many genes are expressed in a mammal has not yet been resolved after the last two REF: 129437 decades of study. There are some direct studies that address patterns of gene expression in different tissues. Mutational load studies (JO Bishop, "The Gene Numbers Game", Cell 2: 81-86 (1974), T. Ohta &M. Kimura, "Functional Organization of Genetic Materials as a Product of Molecular Evolution", Nature 223: 118-119 (1971)) have suggested that there are between 3 x 104 and 105 essential genes. Prior to cDNA cloning techniques, information about the expression of genes comes from RNA complexity studies: analog measurements (measurements in volume) based on observations of populations - mixed RNA molecules with different specificities in abundances. To an unexpected degree, early analog complexity studies have been distorted by hidden complications from the fact that the molecules in each tissue that make up the bulk of their mRNA mass comprise only a small fraction of their total complexity. Finally, cDNA cloning allows digital measurements to be made (ie, sequence-specific measurements in individual species); therefore, the most recent concepts about mRNA expression are based on actual observations of individual RNA species. The brain, liver and kidneys are the mammalian tissues that have been studied most widely by analog RNA complexity measurements. The lowest estimates of complexity are those of Hastie and Bishop (ND Hastie &JB Bishpp, "The Expression of Three Abundance Classes of Messenger RNA in Mouse Tissues", Cell 9: 761-774 (1976)), who have suggested that 26 x 106 nucleotides of the rodent genome are expressed in the brain of 3 x 109 base pairs in the brain, 23 x 10 in the liver and 22 x 106 in the kidney, with an almost complete superposition of sets of RNA. This indicates a very small number of tissue-specific mRNAs. However, experience has shown that these values clearly must be underestimated, because many mRNA molecules, which probably have abundances below the detection limits in this initial study, have been shown to be expressed in brain but they are not detectable in liver or kidney. Many other researchers (JA Bantle &Hahn, "Complexity and Characterization of Polyadenylated RNA in the Mouse Brain", Cell 8: 139-150 (1976); DM Chikaraishi, "Complexity of Cytoplasmic Polyadenylated and Non-Adenylated Rat Brain Ribonucleic Acids ", Biochemistry 18: 3249-3256 (1979)) have measured analogous complexities of between 100-200 x 106 nucleotides in the brain and 2 to 3 times lower estimates in liver and kidney. Of these mRNAs in the brain, 50-65% are not detected in liver or kidney. These values are supported by digital cloning studies (R. J. Milner &J. G. Sutcliffe, "Gene Expression in Rat Brain", Nucí Acids, Res 11: 5497-5520 (1983)).

Analogous measurements in mRNA by volume suggest that the average mRNA length is between 1400-1900 nucleotides. In a systematic digital analysis of brain mRNA length using 200 randomly selected brain cDNAs to measure RNA size by Northern blotting (Milner &Sutcliffe, supra), it has been found that, when data from Size of mRNA are weighted for RNA prevalence, the average length is 1790 nucleotides, the same determined by analog measurements. However, the mRNAs that make up most of the complexity of mRNA in the brain have an average length of 5000 nucleotides. Not only when brain RNAs are rarer, but they tend to be brain-specific, whereas more prevalent brain mRNAs are expressed more ubiquitously and are much shorter on average. These concepts about mRNA lengths have been corroborated more recently from the length of the brain mRNA whose sequences have been determined (JG Sutcliffe, "mRNA in the Mammalian Central Nervous System", Annu .. Rev. Neurosci. 157-198 (1988)). Therefore, the nucleotide complexity 1-2 x 108 and the average mRNA length of 5000 nucleotides calculate an estimated 30,000 mRNA expressed in the brain, of which about 2/3 have not been detected in liver or kidney. The brain apparently constitutes a considerable portion of the tissue-specific genes of mammals. The majority of brain mRNAs are expressed at low concentration. There are no measurements of total mammalian mRNA complexity, nor is it known if the 5000 nucleotides are a length of good mRNA estimated for non-neural tissues. A reasonable estimate of the total number of genes can be between 50,000 and 100,000. What is needed most to advance the chemical understanding of physiological function is a menu of protein sequences encoded by the genome plus the cell types in which each is expressed. To date, protein sequences can be reliably derived only from cDNAs, and not from genes, due to the presence of intervening sequences (introns) in the genomic sequences. Even a complete nucleotide sequence of a mammalian genome will not replace the characterization of its expressed sequences. Therefore, a systematic strategy is needed to collect the transcribed sequences and demonstrate their sites of expression. Such a strategy would be of particular use in the determination of sequences that are differentially expressed within the brain. Necessarily, a final objective of such a study would be to obtain a closure, that is, to identify all the mRNAs. Closure can be difficult to obtain due to the different prevalence of various mRNAs and the large amount of distinct mRNAs that are expressed by many different tissues. The effort to obtain it allows one to obtain a progressively more reliable description of the dimensions of the gene space. The studies carried out in the laboratory of Craig Venter (MD Adams et al., "Commentary DNA Sequencing: Expressed Sequence Tags and Human Genome Project", Science 252: 1651-1656 (1991), MD Adams et al., "Sequence Identification of 2,375 Human Brain Genes", Nature 355: 632-634 (1992)) have resulted in the isolation of randomly chosen cDNA clones from human brain mRNAs, the determination of the short-pass sequences of their 3 'ends, of approximately 300 base pairs, and a compilation of approximately 2500 of these as a database of "expressed sequence labels". This database, although useful, does not provide any knowledge regarding the differential expression. Therefore, it is important to be able to recognize genes based on their total pattern of expression within regions of the brain and other tissues in response to various paradigms, such as the various physiological and pathological states or effects of drug treatment, in instead of simply its expression in a single fabric. Another work has focused on the use of the polymerase chain reaction (PCR) to establish a database. Williams et al. (J.G.K. Williams et al., "DNA Polymorphisms Amplified by Arbitrary Primers Are Useful as Genetic Markers," Nucí Acids Res. 18: 6531-6535 (1990)) and Welsh &; McClelland (J. Wels &McClelland, "Genomic Fingerprinting Using Arbitrarily Primed PCR and a Matrix of Pairwise Combinations of Primers", Nucí Acids Res. 18: 7213-7218 (1990) demonstrated that single primers of 10 units of selected sequences arbitrarily, that is, any 10-unit primer outside the system, when used for PCR with complex DNA templates such as human, plant, yeast or genomic bacterial DNA results in an array of PCR products. it is shown that they involve incomplete complementarity between the primer and template DNA Presumably, poorly matched primer binding sites are partially randomly distributed throughout the genome Occasionally, two of these sites in opposite orientation are located close enough or together to give There is an average of 8-10 products, which vary in size from approximately 0.4 to approximately amin 4 kb and have different mobilities for each primer. The distribution of PCR products show differences between individuals of the same species. These authors propose that unique arbitrary primers can be used to produce information similar to restriction fragment length polymorphism (RFLP) for genetic studies. Other researchers have applied this technology (S.R. Woodward et al., "Random Sequence Oligonucleotide Primers Detect Polymorphic DNA Products Wich Segregate in Inbred Strains of Mice ", Mamm.Genome 3: 73-78 (1992); J. H. Nadeau et al., "Multilocus Markers for Mouse Genome Analysis: PCR Amplification Based on Single of Aribtrary Nucleotide Sequence ", Mamm.Genome 3: 55-64 (1992).) Two groups (J. Welsh et al.," Arbitrarily Primed PCR Fingerprinting of RNA ", Nucí. 20: 4965-4970 (1992); P. Liang &AB Pardee, "Differential Display of Eukaryotic Messenger RNA by Means of the Polymerase Chain Reaction", Science 257: 967-971 (1992)) adapted the method for comparing populations of mRNA In the study by Liang and Pardee, this method, called differential display of mRNA, is used to compare the population of mRNAs expressed by two types of related cells, normal and tumorigenic mouse A31 cells for each experiment, is used a primer of 10 arbitrary units as the 5 'primer and an oligonucleotide complementary to a subset of poly A tails as a 3' anchor primer, performing the PCR amplification in the presence of 35S-dNTP in the cDNAs prepared from two types of cells. cysts are separated in sequenced gels and 50-100 bands ranging from 100-500 nucleotides are observed. The bands probably result from the amplification of the cDNAs corresponding to the 3 'ends of the mRNAs containing the 3' anchor primer complement and a partially mismatched 5 'primer site, as observed in the DNA templates genomic For each primer pair, the pattern of bands amplified from two cDNAs is similar, with intensities of approximately 80% of the bands being indistinguishable. Some of the bands are more intense in one or another of the PCR samples; some were detected only in one of the two samples. Subsequent studies (P. Liang et al., "Distribution and Cloning of Eukaryotic mRNAs by Means of Differential Display: Refinements and Optimization", Nucí Acids Res. 21: 3269-3275 (1993)) have shown that procedural work with low concentrations of input RNA (although it is not quantitative for rarer species), and specificity is mainly received in the last nucleotide of the 3 'anchor primer. At least the third of the differentially detected and identified PCR products corresponds to differentially expressed RNA, with a false positive rate of at least 25%. All of the 50,000 to 100,000 mammalian mRNAs are accessible to this arbitrary primer PCR solution, then approximately 80-95 of the 5 'arbitrary primers and 12 of the 3' anchor primers would be required in approximately 1000 PCR panels and gels for • provide a good probability, calculated by the Poisson distribution, that approximately two-thirds of these mRNAs have been identified.

It is unlikely that all mRNAs are susceptible to detection by this method for the following reasons. For an mRNA to surface in such an examination, it must be sufficiently prevalent to produce a signal on autoradiography and contain a sequence in its 500 nucleotides of the 3 'terminal part capable of serving as a binding site and of prime priming. paired The more prevalent the individual mRNA species is, the more likely it is to generate a product. Therefore, prevalent species can generate bands with many different arbitrary primers. Because this latter property may contain an unpredictable element of opportunity based on the selection of arbitrary primers, it would be difficult to approximate a closure by the arbitrary primer method. In addition, for information to be portable from one laboratory to another and reliable, mismatched priming must be highly reproducible under different laboratory conditions using different PCR machines, with slight variations resulting in reaction conditions. Since the basis for a mismatched priming is little understood, this is a disadvantage for the construction of a database from data obtained by the differential display method of Liang and Pardee. The US patents Numbers 5,459,037 ('037) and 5,807,680 (' 680) describe an improved method of differential display of mRNA species that reduces the uncertainty aspect of the 5 'end generation and allows the data to be absolutely reproducible on different computers. The method does not depend on a potentially paired and potentially reproducible priming, it reduces the number of PCR panels and gels needed for a complete analysis and allows the double-stranded sequence data to accumulate rapidly. In addition, the improved method also reduces the number of concurrent signals that are obtained from the same species of mRNA. Therefore, 'the patents' 037 and' 680 are incorporated by reference, as part of this description. There remains a need for further improvements of the method described in the '037 patent. For example, method specificity can be improved by decreasing poor priming during the synthesis of complementary DNA molecules and during PCR reactions. In addition, the technique can be further refined so that it is more reproducible, more sensitive and easier to use. Preferably, the technique can provide the ability to use sequences obtained to form databases and to explore nucleotide databases such as GenBank to recognize identities and sequence similarities using computer programs such as BLASTN and BLASTX.

BRIEF DESCRIPTION OF THE INVENTION We have developed an improved method for the specific identification of sequence and simultaneous mRNA in a population of mRNA. The improved method classifies the mRNAs based on an identity or direction determined by: 1) a partial nucleotide sequence of length a + b, where a is the length in bases of the recognition site of the restriction endonuclease and b is the number of covered bases, where 6 = Jb = 3, and 2) the distance of this partial sequence from the poly (A) tail. Typically, the identity or direction is determined by a partial sequence that includes a four-base recognition site for a restriction-one endonuclease and four encompassed bases. In a preferred embodiment, the recognition site for a restriction endonuclease is MspI, and the partial sequence is C-C-G-G-N1-N2-N3-N4. Because it depends on the nucleotide sequence of an mRNA and not on its prevalence in a given tissue, the method can consider all of the mRNAs present at concentrations above the detection threshold. In contrast to the differential display and the RAP-PCR methodology, there is no uncertainty aspect for the generation of the 5 'ends. According to a preferred embodiment of the method of the present invention (Figure 1), the cDNA libraries produced from each of the mRNA samples containing copies of the 3 'ends of the end, from the most distal site for MspI at start of the poly (A) tail, or almost all of the poly (A) + mRNA in the initial mRNA sample approximately in accordance with the initial relative concentrations of the mRNA. Because both ends of the inserts for each species are defined exactly by the sequence of the mRNAs themselves, the lengths of fragments are uniform for each species, allowing their subsequent visualization as separate bands on gels. These lengths are constant regardless of the tissue source of the mRNA, an important fundamental concept of the approach. Messenger RNAs lacking the MspI recognition sequences are not represented, but these are relatively rare. These mRNAs are retained by applying the method using a different restriction endonuclease that recognizes a recognition sequence from four different bases. Another aspect of such embodiments of the present invention is the use of sequences adjacent to the 3 'restriction endonuclease site, in a preferred embodiment, an MspI site, to classify the cDNAs into at least two successive PCR steps. The first PCR step uses a primer that anneals the sequences derived from the vector, for example pBC SK +, but extends through CGG from the non-regenerated MspI site to include the first adjacent nucleotide (Nx) of the insert. This stage segregates the initial population of the mRNAs in four subacumulates. In a second PCR stage, each of the four subacumulates produced by the first PCR step is further segregated by dividing it into 64 for a total of 256 subacumulates by using more invasive insert primers (^ N ^^). A fluorescent label is incorporated into the products for detection by laser-induced fluorescence by using fluorescent-labeled 3 'PCR primers in the final PCR step. In a preferred embodiment, a separation technique such as electrophoresis is used to separate the labeled molecules from the PCR product into distinct bands of measurable intensities and corresponding to measurable lengths. Suitable separation techniques include gel electrophoresis, capillary electrophoresis, CLAP, MALD'I spectroscopy and other suitable separation techniques known in the art which are capable of single-base separation over the range of 50-500 bases and which are encompassed by the present invention. In a preferred embodiment, each final PCR reaction product is assigned an identity or address based on a sequence of 8 nucleotides including the four-base restriction endonuclease site plus four recognition bases (eg CCGG-N1-N2-N3- N4) and the distance of this sequence from the junction between the end of the message and the first A of the polyA tail at the 3 'end of the mRNA. When the nucleotide sequence of a PCR product fragment, whether determined experimentally or determined from a database sequence, is known, the fragment is referred to as a digital sequence label (DST): that is, an EST with 3 'end (expressed sequence tag) derived by the method of the present invention. The intensity of the band separated from the labeled PCR product fragments, detected using an appropriate method, preferably laser-induced fluorescence (but radioactive or magnetic labeling can also be used) is quantified and stored for each PCR product fragment. in a database with the address assigned to that PCR product fragment. The intensity of the band separated from the labeled PCR product fragments is proportional to the initial amount of the corresponding mRNA for that PCR product fragment. In general, the method of the present invention comprises: (a) preparing a population of double-stranded cDNA from a population of mRNA using a mixture of anchor primers, each anchor primer having a 5 'terminal part and a part 3 'terminal that includes: (i) a stretch of 7 to 40 waste T; (ii) a site for separation by a first restriction endonuclease that recognizes more than six bases, the site for separation is located towards the 5 'end portion relative to the stretch of the T residues; (iii) a first filler segment of 4 to 40 nucleotides, the first filler segment is located towards the 5 'end portion relative to the site for separation by the first restriction endonuclease; (iv) a second filler segment interposed between the site for separation by a first restriction endonuclease that recognizes more than six bases and the right residue T, and (v) phase adjustment residues that are located in the terminal 3 'part of each of the anchor primers that are selected from the group consisting of -V, -VN, and -VNN, wherein V is a deoxyribonucleotide which is selected from the group consisting of A, C and G, and N is a deoxyribonucleotide that is selected from the group consisting of A, C, G and T, the mixture includes anchor primers that contain all possibilities for V and N; (b) population separation of double-stranded cDNA with the first restriction endonuclease and the second restriction endonuclease, the second restriction endonuclease recognizes a sequence of four nucleotides, to form a population of double-stranded cDNA molecules and a first and second terminal parts, respectively; (c) inserting each double-stranded cDNA molecule of step (b) into a vector in an orientation that is antisense to a bacteriophage-specific promoter within the vector to form a population of constructs containing the inserted cDNA molecules , whereby 5 'and 3' flanking vector sequences are defined adjacent to the 5 'end portion of the direct (sense) strand of the inserted cDNA, and the 3' end portion of the direct strand, respectively, and the constructs have a 3 'flanking vector sequence of at least 15 nucleotides in length between the first restriction endonuclease site and a site defining the initiation of transcription in the promoter; (d) transforming a host cell with the vector into which the separated cDNA has been inserted to produce vectors containing cloned inserts; (e) generating linearized fragments containing the cDNA molecules inserted by digestion of the constructs produced in step (c) with at least one restriction endonuclease that does not recognize sequences either inserted into the cDNA molecules or into the specific promoter of bacteriophage, but sequences are recognized in the vector, so that the resulting linearized fragments have a 5 'flanking vector sequence of at least 15 nucleotides within the 5' vector for the second terminal part of the double-stranded cDNA molecules; (f) 'generating a cRNA preparation of antisense cRNA transcripts by incubating the linearized fragments with a bacteriophage-specific RNA polymerase capable of initiating transcription from the bacteriophage-specific promoter; (g) generating first-strand cDNA by transcribing the cRNA using a reverse transcriptase and an RT 5 'primer that is 15 to 30 nucleotides in length and comprising a nucleotide sequence that is complementary to the sequence of the 5' flanking vector; (h) generating a first set of PCR products by dividing the cDNA from the first chain in a first series of subacumulates and using cDNA from the first chain as templates for a first polymerase chain reaction-with a first PCR primer 3 'of 15 to 30 nucleotides in length which is complementary to the 3' flanking vector sequences between the first restriction endonuclease site and the site defining the initiation of transcription by the bacteriophage-specific promoter and a first 5 'PCR primer. defined with a 3 'terminal part consisting of -Nlf where "N" is one of the four deoxyribonucleotides A, C, G, or T, the first 5' PCR primer is 15 to 30 nucleotides in length and complementary to the 5 'flanking vector sequence with the first complementarity of 5' PCR primers extending into a nucleotide of the specific nucleotides of the cRNA insert, wherein one different from the first PCR primers 'are used in each of the four different subacumulates; (i) generate a second set of PCR products by further dividing the first set of PCR products in each of the first series of sub-cumulatives in a second set of subacumulates and using the first set of PCR products as templates for a second set polymerase chain reaction with a second 3 'PCR primer of 15 to 30 nucleotides in length that is complementary to the 3' flanking vector sequences between the first restriction endonuclease site and the site that defines the initiation of transcription by the promoter bacteriophage specific, and a second 5 'PCR primer defined with a 3' terminal part consisting of Nx Nx, where Nx is identical to the Nx used in the first polymerase chain reaction for that subacumulate, "N" is as in step (h) and "x" is an integer from 1 to 5, the primer is 15 to 30 nucleotides in length and complementary to the 5 'flanking vector sequence with the complementary primer age extending through the insert specific nucleotides of the cRNA at a number of nucleotides equal to "x" +1, wherein a different one of the second 5 'PCR primers are used in different subacumulates of the second series of subacumulates where there are 4X subacumulados in the second series of subacumulados for each of the subacumulados in the first set of subacumulados; (j) separating the second set of PCR products to generate a display of sequence specific products representing the 3 'ends of the mRNAs present in the mRNA population. In a preferred embodiment, a portion of biotin is conjugated to the anchor primers, preferably to the 5 'end portion of the anchor primers. In such an embodiment, the first restricted cDNA is separated from the rest of the cDNAs in step (b) by contacting the first restricted cDNA with a substrate coated with streptavidin. Suitable substrates coated with streptavidin include microtiter plates, PCR tubes, polystyrene spheres, paramagnetic polymer spheres and paramagnetic porous glass particles. A preferred substrate coated with streptavidin is a suspension of paramagnetic polymer spheres (Dynal, Inc., Lake Success, NY). In one embodiment, the 3 nucleotides at the 3 'end of the first 5' PCR primer are linked by phosphodiesterase-resistant bonds, preferably phosphorothioate linkages. In a further embodiment, all 3 nucleotides at the 3 'end of the second 5' PCR primer are linked by phosphodiesterase resistant linkages, preferably phosphorothioate linkages. Preferably, the 3 nucleotides at the 3 'end of both the first and the second 5' PCR primers are linked by phosphorothioate linkages. Typically, one of the primers for the second PCR reaction is conjugated to a fluorescent tag. A suitable fluorescent tag is selected from the group consisting of spiro (isobenzofuran-1 (3H), 9 '- (9H-xanten) -3-one, 6-carboxylic acid, 3', 6'-dihydroxy-6-carboxyfluorescein ( 6-FAM, ABI); Spiro (isobenzofuran-1 (3H), 9 '- (9H) -xanten) -3-one, 5-carboxylic acid, 3', 6'-dihydroxy-5-carboxyfluorescein (5-FAM, Molecular Probes); spiro (isobenzofuran-1 (3H), 9'-xanten) -3-one, 3 ', 6'-dihydroxy-fluorescein (FAM, Molecular Probes); 9- (2,5-dicarboxyphenyl) -3,6-bis (dimethylamino) -xantylium (6-carboxytetramethylrhodamine (6-TAMRA), Molecular Probes); 3, 6-diamino-9- (2-carboxyphenyl) -xantilium (Rhodamine Green "*, Molecular Probes); spiro acid [isobenzofuran-1 (3H), 9'-xanten] -6-carboxylic acid, 5'-dichloro- 3 ', 6' -dihydroxy-2 ', 7'-dimethoxy-3-oxo- (JOE, Molecular Probes); inner salt of (2, 4 -disulf of enyl) -2,3,6,7,12, 13,16, 17-octahydro 1H, 5H, 11H, 15H-xantheno [2,3,4-ij: 5, 6, 7-i 'j'] diquinolizin-8-io, (Texas Red, Molecular Probes); 6- ((4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-propionyl) amino) hexanoic acid (BODIPY FL-X, Molecular Probes); 6- ((4,4-difluoro-1,3-dimethyl-5- (4-methoxyphenyl) -4 -b-ora-3a, 4a-diaza-s-indacen-3-propionyl) amino) hexanoic acid ( BODIPY TMR-X, Molecular Probes); 6- (((4, 4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indacen-3-yl) phenoxy) acetyl) amino) -hexanoic acid (BODIPY TR -X, Molecular Probes); 4, 4-dif luoro-4-bora-3a, 4a-diaza-s-indacen-3-pentanoic acid (BODIPY FL-C5, Molecular Probes); 4, 4-difluoro-5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-propanoic acid (BODIPY FL, Molecular Probes); 4,4-dif luoro-5-phenyl-4-bora-3a, 4a-diaza-s-indacen-3-propionic acid (BODIPY 581/591, Molecular Probes); 4, 4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indacen-3-propionic acid (BODIPY 564/570, Molecular Probes); 4,4-dif luoro-5-styryl-4-bora-3a, 4a-diaza-s-indacen-3-propionic acid; 6- (((4,4-difluoro-5- (2-thienyl) -4-bora-3 a, 4 a-diaza-s-indacen-3-yl) styryloxy) acetyl) aminohexanoic acid (BODIPY 630/650 , Molecular Probes); 6- (((4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indacen-3-yl) styryloxy) acetyl) aminohexanoic acid (BODIPY 650/665, Molecular Probes); and inner salt of 9- (2,4 (or 2,5) -dicarboxyphenyl) -3,6-bis (dimethylamino) -xantilium (TAMRA, Molecular Probes). Other suitable fluorescent labels include 4, 7, 2 ',', 5 ', 7'-hexachloro-6-carboxyfluorescein ("HEX", ABI), "NED" (ABI) and 4, 7, 2', 7 'tetrachlor 6-carboxyfluorescein ("TET", ABI) which are known in the art. Typically, the residues of phase adjustments in step (a) have a 3 'terminal part of -V-N-N. In other embodiments, the phase adjustment residues in step (a) have a 3 'terminal part of -V or -V-N. In a preferred embodiment, the "x" in step (i) is 3. Preferably, the phase adjustment residues in step (a) are -VNN and the "x" in step (i) is 3. Typically , the anchor primers each have 8 to 18 T residues in the waste stream T. In a preferred embodiment, the anchor primers each have 18 T residues in the T waste stream. In other embodiments, each of the anchor primers have from 8 to 18 T residues, preferably from 8 to 16 T residues, more preferably from 8 to 14 T residues, and most preferably from 8 to 12 T residues, in the treatment of residues T. In another preferred embodiment, the anchor primers each have 12 T residues in the waste tract T. Typically, the first anchor primer filler segment is 14 residues in length. In one embodiment, the first filler segment has the nucleotide sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C (SEQ ID NO: 1). In a preferred embodiment, the first filler segment has the nucleotide sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A (SEQ ID NO: 2). Typically, the bacteriophage-specific promoter is selected from the group consisting of the T3 promoter, the T7 promoter and the SP6 promoter. Preferably, the bacteriophage-specific promoter is the T3 promoter. In one embodiment, the primer for priming cDNA transcription from cRNA has the sequence A-G-T-C-G-A-C-G-G-T-A-T-C-G-G (SEQ ID NO: 14). In another embodiment, the primer for priming cDNA transcription from cRNA has the sequence A-G-C-T-C-T-G-T-G-G-T-G-A-G-G-A-T-C (SEQ ID NO: 28). In a further embodiment, the primer for priming transcription of the cDNA from the cRNA has the sequence T-C-G-A-C-T-G-T-G-G-T-G-A-G-C-A-T-G (SEQ ID NO: 35). In one embodiment, the vector is the pBC SK + plasmid separated with Clal and Notl and the 3 'PCR primer in steps (h) and (i) is G-A-G-C-T-C-C-C-C-G-C-G-T (SEQ ID NO: 47).

In another embodiment, the vector is the pBC SK + plasmid separated with Clal and Notl, and the 3 'PCR primer in steps (h) and (i) is G-A-G-C-T-C-G-T-T-T-C-C-C-C-A-G (SEQ ID NO: 48). Typically, the first restriction endonuclease recognizing more than six bases is selected from the group consisting of AscI, Bael, Fsel, Notl, PacI, Prnel PpuMI, RsrII, SapI, SexAI, Sfil, SafI, S rAI, Srfl, Sse83871 and SwaI . A first preferred restriction endonuclease that recognizes more than six bases is Notl. Typically, the second restriction endonuclease that recognizes a sequence of four nucleotiis selected from the group consisting of Mbol, Dpnll, Sau3 AI, Tsp509I, HpalI, Bfal, Csp6I, Msel, Hhal, NalIII, Taal, MspI, MaeII and HinPlI. The second preferred restriction endonucleases that recognize a sequence of four nucleotiare MspI, Sau3 AI and NlalII. Typically, the restriction endonuclease used in step (e) has a nucleotide sequence recognition that incluthe four nucleotide sequence of the second restriction endonuclease used in the step (b) In one embodiment, the second restriction endonuclease is MspI and the restriction endonuclease used in the step (e) is Smal. In another embodiment, the second restriction endonuclease is Taql and the restriction endonuclease used in step (e) is Xhol. In an alternative embodiment, the second restriction endonuclease is HinPlI and the restriction endonuclease used in step (e) is Narl. In another additional embodiment, the second restriction endonuclease is Mae11 and the restriction endonuclease is used in step (e) is AatII. Typically, the vector of step (c) is in the form of a circular DNA molecule having a first and second vector restriction endonuclease sites flanking a vector filler sequence, and further comprising the step of digesting the vector with restriction endonucleases that separate the vector and the first and second restriction endonuclease sites of the vector. Preferably, the vector filler sequence includes an internal vector filler restriction endonuclease site between the first and second restriction endonuclease sites of the vector. A suitable host cell is Escherichia coli. Typically, step (e) includes digestion of the vector with a restriction endonuclease which separates the vector at the internal vector filler restriction endonuclease site. Typically, the restriction endonuclease used in step (e) also separates the vector at the internal vector filler restriction endonuclease site.

For other restriction endonucleases, a general scheme for linearizing a pSK vector without a suitable restriction endonuclease having a six base recognition site containing an internal four base recognition site comprises: (i) dividing the plasmid containing the inserted in two fractions, a first fraction is separated with the restriction endonuclease Xhol, and a second fraction is separated with the restriction endonuclease SalI; (ii) recombining the first and second fractions after separation; (iii) dividing the recombined fractions into thirds and separating the first third with the restriction endonuclease HindIII, the second third with the restriction endonuclease BamHI and the third with the restriction endonuclease EcoRI; and (iv) recombining the thirds after digestion in order to produce a population of linearized fragments of which approximately one sixth of the population corresponds to the separation product for each of the possible combinations of enzymes. Typically, the mRNA population has been enriched from polyadenylated mRNA species. Typically, the separation of the fragments amplified in step (j) is carried out by electrophoresis to receive the products. Preferably, the intensity of the products exhibited after the electrophoresis is approximately proportional to the abundances of the mRNAs corresponding to the products in the original mixture. In a preferred embodiment, the method further comprises a step of determining the relative abundance of each mRNA in the original mixture from the intensity of the product corresponding to that mRNA after electrophoresis. Typically, the step of separating the amplified fragments by polymerase chain fraction by electrophoresis comprises electrophoresis of the fragments in multiple gels. In one embodiment of the invention, the method further comprises the steps of: (k) eluting at least one cDNA corresponding to an mRNA from an electropherogram in which bands representing the 3 'ends of the mRNAs are displayed. present in the sample; (1) amplifying the isolated PCR product in a polymerase chain reaction; (m) cloning the isolated PCR product amplified in a plasmid; (n) producing DNA corresponding to the isolated PCR product cloned from the plasmid; and (o) sequencing the cloned isolated PCR product. Another embodiment of the present invention comprises the steps of: (a) isolating a population of mRNA; (b) preparing a population of double-stranded cDNA from the mRNA population using a mixture of anchor primers, each anchor primer having a 5 'terminal part and a 3' terminal part, and includes: (i) a stretch from 7 to 40 waste T; (ii) a site for separation by a first restriction endonuclease that recognizes more than six bases, the site for separation is located towards the 5 'end portion relative to the stretch of the T residues; (iii) a first filler segment of 4 to 40 nucleotides, the first filler segment is located towards the 5 'end portion relative to the site for separation by the first restriction endonuclease; (iv) a second filler segment interposed between the site for rupture by a first restriction enduclease that recognizes more than six bases and the stretch of the T residues, and (v) phase-adjusting residues -VNN that are located in the 3 'terminal of each of the anchor primers, wherein V is a deoxyribonucleotide which is selected from the group consisting of A, C and G; and N are deoxyribonucleotide which is selected from the group consisting of A, C, G and T, the mixture includes anchor primers which contain all possibilities for V and N; (c) separating the population of double-stranded cDNAs with the first restriction endonuclease and a second restriction endonuclease, the second restriction endonuclease recognizes a sequence of four nucleotides to form a population of double-stranded cDNA molecules having a first and second terminal parts, respectively; (d) inserting each double-stranded cDNA molecule from step (b) into a vector in an orientation that is direct relative to a T3 promoter within the vector to form a population of constructs containing the cDNA molecules inserted, by which defines 5 'and 3' flanking vector sequences adjacent to the 5 'end portion of the inserted cDNA direct strand and the 3' end portion of the direct strand, respectively, and such constructs have a 5 'flanking vector sequence. of at least 15 nucleotides in length between the second restriction-endonuclease site and a site defining the initiation of transcription in the promoter: (e) transforming Escherichia coli with the vector into which the separated cDNA has been inserted to produce vectors containing cloned inserts; (f) generating linearized fragments containing the cDNA molecules inserted by digesting the constructs produced in step (c) with at least one restriction endonuclease that does not recognize sequences in either the cDNA molecules inserted or in the T3 promoter; (g) generating a cRNA preparation of direct cRNA transcripts by incubating the linearized fragments with T3 RNA polymerase capable of initiating transcription from the T3 promoter; (h) generating first-strand cDNA by transcribing the cRNA using a reverse transcriptase and an RT 3 'primer having 15 to 30 nucleotides in length and comprising a nucleotide sequence and which is complementary to the sequence of the 3' flanking vector; (i) generate a first set of PCR products by dividing the cDNA of the first strand in a first series of subacumulates and by using the cDNA of the first strand as templates for a first polymerase chain reaction with a first PCR primer 3 'of 15 to 30 nucleotides in length which is complementary to the 3' flanking vector sequences, which are 3 'to the first restriction endonuclease site and a first 5' PCR primer defined with a 3 'terminal part consisting of of N1 (where "N" is one of the four deoxyribonucleotides A, C, G or T, the first 5 'PCR primer is 15 to 30 nucleotides in length and is complementary to the 5' flanking vector sequence with the first complementary 5 'PCR primer extending within a nucleotide of the specific nucleotides of cRNA insert, wherein a different one of the first 5' PCR primers is used in each of the four different subacculates; (j) generate a second set of PCR products by further dividing the first set of PCR products in each of the first series of sub-cumulatives in a second set of subacumulates and using the first set of PCR products as templates for a second set polymerase chain reaction with a second 3 'PCR primer of 15 to 30 nucleotides in length which is complementary to the 3' flanking vector 3 'to the first restriction endonuclease site and a second 5' PCR primer defined by a terminal part 3 'consisting of Nx Nx, where x is identical to the Nx used in the first polymerase chain reaction for this subacumulate, "N" is as in stage (i), and "x" is a number whole which is selected from the group consisting of 3 and 4, the primer is 15 to 30 nucleotides in length and is complementary to the 5 'flanking vector sequence with primer complementarity extending through ro of the insert specific nucleotides of the cRNA in a number of nucleotides equal to "x" = 1, wherein a different one of the second 5 'PCR primers is used in different subacumulates of the second series of subacumulates and where there are 4X subacumulados in the second series of subacumulados; (k) separating the second set of PCR products to generate a display of the sequence specific products representing the 3 'ends of the mRNAs present in the mRNA population. Typically, the mixture of 48 anchor primers has the sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 5). In a preferred embodiment, the mixture of 48 anchor primers has the sequence G-A-T-T-T-C-A-A-C-T-G-G-A-A-G-C-G-C-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 8). Typically, the mixture of 12 anchor primers has the sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 4). In a preferred embodiment, the mixture of 12 anchor primers has the sequence G-A-T-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-C-G-C-A-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 7). Typically, the mixture of 3 anchor primers has the sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 3). In a preferred embodiment, the mixture of 3 anchor primers has the sequence G-A-T-T-T-C-A-A-C-T-G-G-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 6).

In a preferred embodiment, the first restriction endonuclease is MspI and the second restriction endonuclease is Notl. Typically, the first 5 'PCR primer is G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 22). In a preferred embodiment, the 3 'PCR primer in the second polymerase chain reaction is the nucleotide of SEQ. FROM IDENT. NO: 47 conjugated to a fluorescent tag, more preferably, the nucleotide of SEC. FROM IDENT. NO: 47 conjugated to 6-FAM. The appropriate values of "x" in step (i) are integers from 1 to 5. Preferably, "x" in step (i) is 3. Typically, a method to detect a change in mRNA expression pattern in a tissue associated with a physiological or pathological change, it comprises the steps of: (a) obtaining a first sample of normal or neoplastic tissue that is subjected to physiological or pathological change; (b) isolating a population of mRNA from the first sample; (c) determining the pattern of mRNA expression in the first tissue sample by performing steps (a) - (j) of the general method to generate a first display of the sequence specific products representing the 3 'ends of the mRNAs present in the first sample; (d) obtain a second tissue sample that has undergone physiological or pathological change; (e) isolating a population of mRNA from the second sample; (f) determining the pattern of mRNA expression in the second tissue sample by performing steps (a) - (j) of the general method to generate a second display of the sequence specific products representing the 3 'ends of the mRNAs present in the second sample; and (g) comparing multiple displays to determine the effect of physiological or pathological change on the pattern of mRNA expression in the tissue. Typically, more than two samples are compared. In the preferred embodiments, 3, more preferably at least 4, samples are taken at multiple times, and compared. Typically, the physiological or pathological change is selected from the group consisting of Alzheimer's disease, parkinsonism, ischemia, alcohol addiction, drug addiction, schizophrenia, amorphic lateral sclerosis, multiple sclerosis, depression and manic-depressive bipolar disorders. Typically, physiological or pathological change is associated with learning or memory, emotion, neurotoxicity to glutamate, eating behavior, smell, vision, movement disorders, viral infection, electrochock therapy, administration of a drug or effects toxic collateral drugs. Typically, the physiological or pathological change is selected from the group consisting of circadian variation, aging and long-term potentiation. In general, the physiological or pathological change is selected from processes mediated by transcription factors, intracellular second messengers, hormones, neurotransmitters, growth factors and neuromodulators. Alternatively, the physiological or pathological change is selected from processes mediated by cell-cell contact, cell-substrate contact, cell-extracellular matrix contact and cell membrane-cytoskeleton contact. Preferably, normal or neoplastic tissue comprises cells that are taken or derived from an organ or system of organs that are selected from the group consisting of the cardiovascular system, the lymphatic system, the respiratory system, the digestive system, the peripheral nervous system , the central nervous system, the enteric nervous system, the endocrine system, the integument (which includes skin, hair and nails), the skeletal system (which includes bone and muscle, the urinary system and the reproductive system. Normal or neoplastic tissue comprises cells that are taken or that are derived from the group consisting of epithelium, endothelium, mucosa, glands, blood, lymph, connective tissue, cartilage, bone, smooth muscle, skeletal muscle, cardiac muscle, neurons, glia, spleen, thymus, hypophysis, thyroid, parathyroid, adrenal cortex, adrenal medulla, adrenal cortex, pineal, skin, hair, nails, teeth, liver or, pancreas, lung, kidney, bladder, urethra, breast, ovary, uterus, vagina, testes, prostate, penis, eye and ear. Typically, normal or neoplastic tissue is derived from a structure within the central nervous system that is selected from the group consisting of the retina, cerebral cortex, olfactory bulb, thalamus, hypothalamus, anterior pituitary, posterior pituitary, hippocampus, nucleus, amygdala , striago, cerebellum, brainstem, supraiasmatic nucleus, and spinal cord. Typically, a method for detecting a difference in the action of a medicament to be examined and a known compound comprises the steps of: (a) obtaining a first tissue sample from an organism treated with a compound of known physiological function; (b) isolating a population of mRNA from the first sample; (c) determining the pattern of mRNA expression in the first tissue sample by performing steps (a) - (j) of the general method to generate a first display of sequence specific products representing the 3 'ends of the mRNAs present in the first sample; (d) obtaining a second tissue sample from an organism treated with a drug to be examined to determine a difference in the action of the drug and the known compound; (e) isolating a population of mRNA from the first sample; (f) determining the mRNA expression pattern in the second tissue sample by performing steps (a) - (j) of the general method to generate a second display of sequence specific products representing the 3 'ends of the mRNAs present in the second sample; and (g) comparing the first and second exhibits in order to detect the presence of mRNA species whose expression is not affected by the known compound but is affected by the drug to be examined, so a difference is indicated in the action of the drug to be examined and the known compound. Typically, the medicament to be examined is selected from the group consisting of antidepressants, neuroleptics, tranquilizers, anticonvulsants, monoamine oxidase inhibitors, stimulants, anti-parkinsonism agents, skeletal muscle relaxants, relaxants, local anesthetics, cholinergics, antiviral agents, antispasmodics, steroids and non-spheroidal anti-inflammatory drugs. More generally, the terms "medicament to be examined" and "medicament to be tested" are used herein to refer to a broad class of useful chemical and therapeutic agents that include physiologically active steroids, antibiotics, antifungal agents, antibacterial agents, antineoplastic agents, analgesics and combinations of analgesics, anorexics, anthelmintics, antiarthritics, anti-asthma agents, anticonvulsants, antidepressants, antidiabetic agents, antidiarrheal agents, antihistamines, anti-inflammatory agents, preparations against migraine, preparations against discomfort due to movement, elements against nausea, antiparkinsonian drugs, ant iprur? icos, antipsychotics, antipyretics, antispasmodics that include gastrointestinal and urinary; anticholinergics, sympathetic mimetics, xanthine derivatives, cardiovascular preparations including calcium channel blockers, beta blockers, antiarrhythmics, antihypertensive diuretics, vasodilators including general, coronary, peripheral and cerebral; stimulants of the central nervous system, preparations against cough and cold, decongestants, hormones, hypnotics, immunosuppressants, muscle relaxants, parasympatholytic, parasimpat icomimetics, psychotics, sedatives, tranquilizers, allergens, antihistamines, anti-inflammatory agents, peptides and physiologically active proteins, agents that function as filters against ultraviolet radiation, perfumes, insect repellents, hair dyes and the like. The term "physiologically active" in describing the agents contemplated herein is used in a broad sense to encompass not only those agents that have a direct pharmacological effect on the host but also those that have an indirect or observable effect which is useful in the medical arts, for example, the generation of tissue color or opacification for diagnostic purposes, ultraviolet radiation filtration of tissues and the like. For example, typical fungistatic and fungicidal agents include thiabendazole, chloroxine, amphotericin, candicidin, fungimycin, nystatin, chlordantoin, clotrimazole, etonam nitrate, miconazole nitrate, pyrrolonitrile, salicylic acid, fezationa, ticlaton, tolnaftate, triacetin, zinc, pyrithione and sodium pyrithione. Steroids include cortisone, shortdoxone, fluoracetonide, fludrocortisone, difluorsone diacetate, flurandrenolone acetonide, medrisone, amcinafel, amcinafide, betamethasone and its esters, chlorprednisone, chlortelone, descinolone, desonido, dexame asone, dichlorisone, difluoprednate, lucloronide, flumethasone, flunisolide, fluocinonide, flucortolone, fluorometalone, fluperolone, fluprednisolone, meprednisone, methylmeprednisone, parametasone, prednisolone, and predisone. Antibacterial agents include sulfonamides, penicillins, cephalosporins, penicillinase, erythromycins, linomycins, vancomycins, tetracyclines, chloramphenicol, streptomycins and the like. Specific examples of antibacterials include erythromycin, erythromycin ethyl carbonate, erythromycin estolate, erythromycin glycetate, erythromycin ethylsuccinate, erythromycin lactobionate, lincomycin, clindamycin, tetracycline, chlortetracycline, demeclocycline, doxycycline, metacycline, oxytetracycline, minocycline, and the like. Peptides and proteins include, in particular, small to medium peptides, eg insulin, vasopressin, oxytocin, growth factors, cytokines as well as larger proteins such as human growth hormone. Other agents include various therapeutic agents such as xanthines, triamterene and theophylline, the antitumor agents 5-f 1 uor our idi nade s ox irri bó si do, 6-mercaptopurine deoxyriboside, vidarabine, the narcotic analgesics hydromorphone, cyclazine, pentazocine, bupomorphine, compounds containing oxalic anions, heparin, prostaglandins and compounds similar to prostaglandin, cromolyn sodium, carbe noxo 1 ona, polyhydroxy compounds, dopamine, dobutamine, 1-dopa, α-methyldopa, angiotensin antagonists, polypeptides such as bradykinin, insulin, adrenocorticotrophic hormone (ACTH), enkephalins, endorphins, somatostatin, secretin and various compounds such as tetracyclines, bromocriptine, lidocaine, cimetidine or any related compound. Other agents include iododeoxyuridine, podophyllin, theophylline, isoprot erenol, acetone of trame inolone, hydrocortisone, indomethacin, butazone, paraminobenzoic acid, aminopropionitrile and penicillamine. The aforementioned list is by no means intended to be exhaustive and any physiologically active agent may be tested by the method of the present invention. Typically, a database comprising the data produced by the quantification of the display of sequence-specific PCR products is constructed. Typically, the database further comprises data regarding sequence relationships, gene mapping and cell distributions. In one embodiment, the invention provides a method for recognizing sequence identities and similarities between the sequences of the 3 'ends of the mRNA molecules present in a sample and a sequence database, comprising the steps of: (a) preparing a population of double-stranded cDNA from a population of mRNA using a mixture of anchor primers, each anchor primer having a 5 'terminal and a 3' terminal part and includes: (i) a stretch of 7 to 40 waste T; (ii) a site for separation by a first restriction endonuclease that recognizes more than six bases, the site for separation is located toward the 5 'end portion relative to the T residue portion; (iii) a first filler segment of 4 to 40 nucleotides, the first filler segment is located towards the 5 'end portion relative to the site for separation by the first restriction endonuclease; (iv) a second filler segment interposed between the site for separation by a first restriction endonuclease that recognizes more than six bases and the stretch for residues T, and (v) phase adjustment residues that are located at the 3 'end part of each of the anchor primers that are selected from the group consisting of -V, -VN, and -VNN, wherein V is a deoxyribonucleotide that is selected from the group consisting of A, C and G; and N is a deoxyribonucleotide which is selected from the group consisting of A, C, G and T, the mixture includes anchor primers containing all possibilities for V and N; (b) separating the population of double-stranded cDNAs with the first restriction endonuclease and a second restriction endonuclease, the second restriction endonuclease recognizes a sequence of four nucleotides to form a population of double-stranded cDNA molecules having a first and second terminal parts, respectively; (c) inserting each double-stranded cDNA molecule of step (b) into a vector in an orientation that is antisense to the bacteriophage-specific promoter within the vector to form a population of constructs containing the inserted cDNA molecules, whereby they define 5 'and 3' flanking vector sequences adjacent to the 5 'end portion of the direct (sense) strand of the inserted cDNA, and the 3' end portion of the direct strand, respectively, and the constructs have a 3 'flanking vector sequence of at least 15 nucleotides in length between the first restriction endonuclease site and a site defining the start of transcription in the promoter; (d) transforming a host cell with the vector into which the separated cDNA has been inserted, to produce vectors containing cloned inserts; (e) generating linearized fragments containing the cDNA molecules inserted by digesting the constructs produced in step (c) with at least one restriction endonuclease that does not recognize sequences in either the cDNA molecules inserted or in the specific promoter of bacteriophage, but which recognizes sequences in the vector, such that the resulting linearized fragments have a 5 'flanking vector sequence of at least 15 nucleotides within the 5' vector relative to the second terminal part of the chain cDNA molecules double; (f) generating a cRNA preparation of antisense cRNA transcripts by incubating the linearized fragments with a bacteriophage-specific RNA polymerase capable of initiating transcription from the bacteriophage-specific promoter; '(g) generating first-strand cDNA by transcribing the cRNA using a reverse transcriptase and a 5' RT primer having 15 to 30 nucleotides in length and comprising a nucleotide sequence which is complementary to the 5 'flanking vector sequence; (h) generating a first set of PCR products by dividing the cDNA of the first strand in a first set of subacumulates and using the cDNA of the first strand as templates for a first polymerase chain reaction with a first PCR primer. 'from 15 to 30 nucleotides in length which is complementary to the 3' flanking vector sequences between the first restriction endonuclease site and the site defining the initiation of transcription by the bacteriophage-specific promoter and a first 5 'PCR primer. defined with a 3 'terminal part consisting of Nx, where "N" is one of four deoxyribonucleotides A, C, G or T, the first 5 'PCR primer has 15 to 30 nucleotides in length and is complementary to the 5' flanking vector sequence with the first PCR primers 5 'of complementarity extending within a nucleotide of the insert specific nucleotides of the cRNA, wherein a different one of the first 5' PCR primers is used in each of the four different subacculates; (i) generate a second set of PCR products by further dividing the first set of PCR products in each of the first series of sub-cumulatives in a second set of subacumulates and using the first set of PCR products as templates for a second set polymerase chain reaction, with a second 3 'PCR primer of 15 to 30 nucleotides in length that is complementary to the 3' flanking vector sequences between the first restriction endonuclease site and the site defining the start of transcription by a specific bacteriophage promoter, and a second 5 'PCR primer defined with a 3' terminal part consisting of N. Nx, where N1 is identical to the x used in the first polymerase chain reaction for that subaqueous ulated, "N" is as in step (h) and "x" is an integer from 1 to 5, the primer is 15 to 30 nucleotides in length and complementary to the 5 'flanking vector sequence with the primer complementarity extending through the specific nucleotides of the cRNA insert in a number of nucleotides equal to "x" +1, wherein a different one of the second 5 'PCR primers are used in different subacumulates of the second series of subacumulates where there are 4X subacumulados in the second series of subacumulados for each of the subacumulados in the first set of subacumulados; (j) separating the second set of PCR products to generate a display of sequence specific products representing the 3 'ends of the mRNAs present in the mRNA population. (k) eluting at least one cDNA corresponding to a MRNA from an electropherogram in which bands representing the 3 'ends of the mRNA present in the sample are displayed, - (1) amplifying the cDNA eluted in a polymerase chain reaction; (m) cloning the amplified cDNA in a plasmid; (n) producing DNA corresponding to the cloned DNA of the plasmid; (o) determining the sequence of the cloned cDNA; (p) determining the corresponding nucleotide sequences from a database of the nucleotide sequences, the corresponding nucleotide sequences are bounded by the most distal recognition site for the second endonuclease and the start of the poly (A) tail; and (q) comparing the sequence of the cloned cDNA with the corresponding nucleotide sequences whereby identities and sequence similarities are recognized between the sequence of the 3 'ends of the mDNA molecules present in a sample and a database of the sequences . Typically, the method further comprises the step of: (r) comparing the length and quantity of the PCR products in a two-dimensional graphical display. In general, the method also comprises the steps of: (s) determining the expected length of the corresponding nucleotide sequence, which is equal to the sum of the lengths of the corresponding nucleotide sequence determined from the database, the length , of the 5 'PCR sequence hybridizable to the vector sequence, the length of the remaining anchor primer sequence, an intermediate segment of the vector sequence and the length of the 3' PCR sequence that can be hybridized with the sequence of the vector; and (t) comparing the length of the PCR product with the determined expected length of the corresponding nucleotide sequence, wherein the expected length of the corresponding nucleotide sequence is indicated by the two-dimensional graphical display by use of a graphic symbol or text character . Suitable graphic symbols include vertical and horizontal lines, intersection of short line segments such as crosses and "x" and geometric shapes that include circles and polygons. In another embodiment, the invention provides a method for recognizing sequence identities and similarities between the sequence of a cDNA fragment corresponding to a mRNA molecule present in a sample, and a sequence database, comprising the steps of: elute a cDNA fragment corresponding to a mRNA molecule present in a sample; amplifying the cDNA fragment eluted in a polymerase chain reaction to produce an amplified cDNA fragment; cloning the amplified cDNA fragment into a plasmid; producing a DNA molecule corresponding to the cloned cDNA fragment; sequencing the produced DNA molecule, whereby the sequence of the eluted cDNA fragment is determined; and comparing the sequence of the cDNA fragment eluted with the sequences in a database whereby the identities and similarities of the sequence are recognized. Typically, the step of comparing the sequence of the cDNA fragment eluted with the sequences in a database is performed using a computer. Typically, the method also comprises the additional step of graphically displaying the results of the comparison. In general, the identities and sequence similarities between the sequence of a cDNA fragment corresponding to a mRNA molecule present in a sample and a sequence database are recognized by a method comprising the steps of: eluting a fragment of CDNA corresponding to a mRNA molecule present in a musetra, wherein the cDNA fragment has a length determined by the position of a restriction endonuclease recognition site and a poly (A) tail of the mRNA molecule; determining a partial sequence of the cDNA fragment by performing a polymerase chain reaction with a 5 'PCR primer corresponding to the sequence of the restriction endonuclease recognition site, and comparing the determined partial sequence of the eluted cDNA fragment and the length of the cDNA fragment with the sequences in a database so that identities and sequence similarities are recognized. In another embodiment, the present invention provides a method for producing a transformed polynucleotide sequence database entry, comprising the steps of: choosing a source sequence from a polynucleotide sequence database entry; locate a poly (A) tail sequence within the source sequence; locating a sequence of endonuclease recognition site within the source sequence that is closest to the first recognition site; determining an index sequence consisting of about two to about six nucleotides adjacent to the endonuclease recognition site; determining a correlation sequence within the source sequence, the correlation sequence includes the sequence linked to the poly (A) tail and the endonuclease recognition site and including at least part of the endonuclease recognition site, - determining the length of the correlated sequence; and storing information regarding the location and sequence of the poly (A) tail, the location and sequence of the endonuclease recognition site, and the length of the correlation sequence in relation to the source sequence, thereby producing a Transformed database entry. Typically, the method includes the step of graphically displaying the length of the correlated sequence in relation to the index sequence. Preferably, the restriction endonuclease is selected from the group consisting of MSPI, Taql and HinPlI. The invention also provides a method for improving the resolution of the length and quantity of PCR products by decreasing the background that is due to amplification of the non-targeted cDNAs, comprising the steps of: selecting a sample from a population of cRNA, wherein each cRNA molecule comprises an insert sequence and a vector-derived sequence; perform reverse transcription using a reverse transcription primer that hybridizes with a vector-derived sequence that extends from about five nucleotides to about six nucleotides within the insert sequence to produce a reverse transcription product of cDNA; subdivide the reverse transcription product of cDNA; performing at least one polymerase chain reaction using the subdivided cDNA reverse transcription product, a 3 'PCR primer and a 5' PCR primer that hybridizes to the vector-derived sequence and extends from about 7 nucleotides to about 9 nucleotides within the insert sequence to produce a PCR product, whereby the background is decreased which is due to amplification of the non-targeted cDNAs.

Typically, there are sixteen accumulated reverse transcription reactions and there are sixteen different reverse transcription primers. In general, there are 4X subacumulated polymerase chain reactions, where X is the difference between the number of nucleotides in which the 5 'PCR primer extends within the insert sequence, and the number of nucleotides that the primer extends of reverse transcription within the insert sequence.

BRIEF DESCRIPTION OF THE DRAWINGS These and other features, aspects and advantages of the present invention will be better understood with reference to the following description, appended claims and accompanying drawings, in which: Figure 1 is a diagrammatic representation of the improved method of the present invention showing the various priming, cutting, cloning, transcription of antisense RNA and amplification sequences showing the anchors and other primers schematically see text for complete sequences; Figure 2 is a diagrammatic representation of one embodiment of the improved method using biotinylated anchor primers with substrate coated with esptreptavidin and showing the various priming, cutting, cloning, transcription steps of antisense and amplification RNA showing the anchor sequences and other primers schematically see text for complete sequences; Figure 3 is a graph of the relative abundance of labeled PCR products versus product length, in base pairs, using a fluorescent detection system, which shows analysis of PCR products that are obtained using a 5 'PCR primer. CGACGGTATCGGGGTG (SEQ ID NO: 42), from mRNA samples of human MG63 cells lacking serum (A) or with serum (B), the data of (A) and (B) are superimposed on the panel ( C) lower using software for comparison of the relative expression levels between samples, - Figure 4 is a graph comparing the relative abundance of the labeled PCR products versus product length in base pairs using a fluorescent detection system for the method using two steps of PCR vesrus the method using only one PCR step, which shows the results that are obtained from the analysis of mRNA extracted from MG63 osteosarcoma cells lacking their ero (A and C) or with serum (B and D) using either a PCR step (A-D) or two steps of PCR (E-H), presenting data from PCR primers 5 '109T (C-G-A-C-G-G-T-A-T-C-G-G-T-G-C-A, (SEQ ID NO: 43) and 45A (CGACGGTATCGGAGCA, ID SECTION NO: 44), which differ only in the NI position (in bold), for samples lacking serum (os-) or with serum (os +), which shows that the PCR products generated with 109T and 45A appear to be almost identical to the templates produced by the one-step PCR (AD) method, while the products detected following PCR from templates produced using the two-stage PCR method is altogether very different (EH); Figure 5 is a graph comparing the relative abundance of the labeled PCR products versus the product length, in base pairs using a fluorescent detection system to compare the results obtained using the standard method depicted in Figure 1 and the method of magnetic sphere method shown in Figure 2, which shows that the data of the magnetic sphere mode show a remarkable increase in the capacity of reproduction through the samples (similarity of fragments generated and consistency of intensity values) in comparison with the data derived from the standard modality of the method; Figure 6 is a graph showing the linear relationship between the cRNA concentration and the peak amplitude of the resulting PCR product for various different tissues; Figure 7 shows the nucleotide sequences and restriction maps of the multiple cloning sites of the plasmids pBC SIC / DGT1, pBS SK "/ DGT2, pBS SK * / DGT3, pBC SK + / DGT4 and pBS SK + / DGT5; and Figure 8 is a diagrammatic representation of the improved method using biotinylated anchor primers with streptavidin coated substrate and showing the various steps of priming, separation, cloning, direct RNA transcription and amplification showing the anchor sequences and other primers schematically see text for complete sequences.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES We have developed an improved method for simultaneous specific identification of sequence and mRNA display in a mRNA population which has a number of applications in the determination of drug action mechanisms, drug testing, the study of physiological and pathological conditions and genomic mapping. The improved method and its applications are discussed in the following.

I. SPECIFIC SIMULTANEOUS IDENTIFICATION OF SEQUENCE OF mRNA A method according to the present invention, based on the polymerase chain reaction (PCR) technique, provides a means for viewing almost every mRNA expressed by normal or neoplastic cells or eukaryotic tissue as a distinct band on a gel whose intensity corresponds in a general way to the concentration of the mRNA. The method is based on the observation that virtually all mRNAs conclude with a 3 '-poly (A) tail, but do not rely on the binding specificity of primer to tail. The improved method is illustrated schematically in three embodiments in Figures 1, 2 and 8. In general, the improved method comprises: (a) preparing a population of double-stranded cDNA from a population of mRNA using a mixture of primers of anchor, each anchor primer has a 5 'terminal part and a 3' terminal part and includes: (i) a stretch of 7 to 40 T residues; (ii) a site for separation by a first restriction endonuclease that recognizes more than six bases, the separation site is located towards the 5 'end portion in relation to the waste site T; (iii) a first filler segment of four to 40 nucleotides, the first filler segment is located towards the 5 'end portion relative to the site for separation by the first restriction endonuclease; (iv) a second filler segment interposed between the site for cleavage by a first restriction endonuclease that recognizes more than six bases and the tract of residues T, and (v) phase adjustment residues located in the terminal 3 'part of each one of the anchor primers which is selected from the group consisting of -V, -VN, -VNN, preferably -VNN, wherein V is a deoxyribonucleotide which is selected from the group consisting of A, C and G; and N is a deoxyribonucleotide which is selected from the group consisting of A, C, G and T, the mixture includes anchor primers containing all possibilities for V and N; (b) separate the population of double-stranded cDNA with the first restriction endonuclease and the second restriction endonuclease, the second restriction endonuclease recognizes a sequence of four nucleotides, to form a population of double-stranded cDNA molecules have a first and second terminal parts, -respectively; (c) inserting each double-stranded cDNA molecule of step (b) into a vector in an orientation that is antisense to a bacteriophage-specific promoter within the vector to form a population of constructs containing the inserted cDNA molecules , whereby 5 'and 3' flanking vector sequences are defined adjacent to the 5 'end portion of the direct (sense) strand of the inserted cDNA and the 3' end portion of the direct strand, respectively, and the constructs have a 3 'flanking vector sequence of at least 15 nucleotides in length between the first restriction endonuclease site and a site defining the initiation of transcription in the promoter; (d) transforming a host cell with the vector into which the separated cDNA has been inserted to produce vectors containing cloned inserts; (e) generating linearized fragments containing the cDNA molecules inserted by digestion of the constructs produced in step (c) with at least one restriction endonuclease that does not recognize sequences either inserted into the cDNA molecules or into the specific promoter of bacteriophage, but sequences are recognized in the vector, so that the resulting linearized fragments have a 5 'flanking vector sequence of at least 15 nucleotides within the 5' vector for the second terminal part of the double-stranded cDNA molecules; (f) generating a cRNA preparation of antisense cRNA transcripts by incubating the linearized fragments with a bacteriophage-specific RNA polymerase capable of initiating transcription from the bacteriophage-specific promoter; (g) generating first-strand cDNA by transcribing the cRNA using a reverse transcriptase and an RT 5 'primer that is 15 to 30 nucleotides in length and comprising a nucleotide sequence that is complementary to the sequence of the 5' flanking vector; (h) 'generate a first set of PCR products by dividing the cDNA of the first strand in a first series of subacumulates and using the cDNA of the first strand as templates for a first polymerase chain reaction with a first PCR primer 3 'of 15 to 30 nucleotides in length which is complementary to the 3' flanking vector sequences between the first restriction endonuclease site and the site defining the initiation of transcription by the bacteriophage-specific promoter and a first 5 'PCR primer. defined with a 3 'terminal part consisting of Nl; wherein "N" is one of the four deoxyribonucleotides A, C, G, or T, the first 5 'PCR primer is 15 to 30 nucleotides in length and is complementary to the 5' flanking vector sequence with the first complementary of 5 'PCR primers extending into a nucleotide of the specific nucleotides of the cRNA insert, wherein a different one of the first 5' PCR primers is used in each of the four different subacculates; (i) generate a second set of PCR products by further dividing the first set of PCR products in each of the first series of sub-cumulatives in a second set of subacumulates and using the first set of PCR products as templates for a second set polymerase chain reaction with a second 3 'PCR primer of 15 to 30 nucleotides in length that is complementary to the 3' flanking vector sequences between the first restriction endonuclease site and the site that defines the initiation of transcription by the promoter bacteriophage specific, and a second 5 'PCR primer defined with a 3' terminal part consisting of Nx Nx, where Nx is identical to N-, used in the first polymerase chain reaction for that subacumulate, "N" is as in step (h) and "x" is an integer from 1 to 5, the primer is 15 to 30 nucleotides in length and complementary to the 5 'flanking vector sequence with the complementary of primer extending through the insert specific nucleotides of the cRNA at a number of nucleotides equal to "x" +1, wherein a different one of the second 5 'PCR primers are used in different subacumulates of the second series of subacumulates where there are 4X subacumulados in the second series of subacumulados for each of the subacumulados in the first set of subacumulados; and (j) separating the second set of PCR products to generate a display of sequence specific products representing the 3 'ends of the mRNAs present in the mRNA population. In an alternative embodiment, step (c) above comprises inserting each double-stranded cDNA molecule of step (b) into a vector in an orientation that is direct relative to the bacteriophage-specific promoter within the vector to form a population of constructs containing the cDNA molecules inserted (figure 8).

A. Preparation of double-stranded cDNA The first step in the method requires a population of mRNA. Methods of RNA extraction are well known in the art and are described, for example, in J. Sambrook et al., "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory Press, Cois Spring Harbor, New York, 1989). , vol. 1, chapter 7, "Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells", incorporated herein as this reference. Other methods of isolation and extraction are also well known. Typically, the isolation is carried out in the presence of chaotropic agents such as guanidinium chloride or guanidinium thiosionate, although other detergents and extractants can alternatively be used. Typically, the mRNA is isolated from the total extracted RNA by chromatography on oligo (dT) -cellulose or other chromatographic medium that has the ability to bind the 3 'polyadenylated portion of the mRNA molecules. Alternatively, although less preferable, total RNA can be used. However, it is generally preferred to isolate poly (A) RNA. " The double-stranded cDNAs are then prepared from the mRNA population using a mixture of anchor primers to initiate reverse transcription. Each anchor primer has a 5 'terminal part and a 3' terminal part and includes: (i) a stretch of 7 to 40 T residues; (ii) a site for separation by a first restriction endonuclease that recognizes more than six bases, the site for separation is located towards the 5 'end portion relative to the stretch of the T residues; (iii) a first filler segment of 4 to 40 nucleotides, the first filler segment is located towards the 5 'end portion relative to the site for separation by the first restriction endonuclease; (iv) a second filler segment interposed between the site for separation by a first restriction endonuclease that recognizes more than six bases and the tract of T residues, and (v) phase adjustment residues that are located in the 3 'terminal part of each of the anchor primers that are selected from the group consisting of -V, -VN, and -VNN, wherein V is a deoxyribonucleotide that is selected from the group consisting of A, C and G; and N is a deoxyribonucleotide which is selected from the group consisting of A, C, G and T, the mixture includes anchor primers that contain all possibilities for V and N where the phase adjustment residues in the mixture are defined by one of -V, -VN, or -VNN. Where the anchor primers have phase adjustment residues of -V, the mixture comprises a mixture of three anchor primers. When the anchor primers have phase adjustment residues of -V-N, the mixture comprises a combination of twelve anchor primers. When the anchor primers have phase adjustment residues of -V-N-N, the mixture comprises a combination of 48 anchor primers. Typically, the anchor primers each have 18 T residues in the waste section T, ending in -V-N-N, and having a first filler segment of 14 residues in length. Preferred sequences of the first filler segment are selected from the group consisting of A-A-C-T-G-G-A-A-G-A-T-T-C (SEQ ID NO: 1) and G-A-T-T-C-A-A-C-T-G-G-A-A (SEQ ID NO: 2). Typically, the para-separation site by a restriction endonuclease that recognizes more than six bases is the Notl cleavage site. A preferred set of three anchor primers have the sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 3). Another preferred set of twelve anchor primers has the sequence A-A-C-T-G-G-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 4). A further preferred set of 48 anchor primers have the sequences A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 5).

In a pre-determined manner, the conjugate of 3 anchor primers has the sequence G-A-T-T-T-T-C-A-C-T-G-G-A-A-G-C-G-C-C-C-C-C-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 6). In another preferred embodiment, the set of 12 anchor primers have the sequence G-A-A-T-T-C-A-A-C-T-G-G-A-G-C-G-G-C-C-C-C-G-C-A-G-G-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 7). In an especially preferred embodiment, the set of 48 anchor primers have the sequence G-A-T-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 8). A member of this mixture of anchor primers initiates synthesis at the fixed position at the 3 'end of all copies of a species of mRNA in the sample, thereby defining a final 3' point for each species. This reaction is carried out under conditions for the preparation of double-stranded cDNA from mRNA, as is well known in the art. Such techniques are described, for example, in Volume 2 of J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", entitled "Construction and Analysis of cDNA Libraries." Suitable transcriptases include avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV). A preferred reverse transcriptase is the reverse transcriptase of MMLV.

In preferred embodiments of the invention, magnetic spheres are used to improve the preparation of the cDNA population (Figures 2 and 8). Typically, the biotin portion is conjugated to the 5 'end portion of the anchor primer and the first restricted cDNA is separated from the rest of the DNA by contacting the first restricted cDNA with a streptavidin coated substrate, such as a number of magnetic spheres. coated with streptavidin.

B. Separation of the cDNA sample with restriction endonucleases The cDNA sample is prepared with two restriction-endonucleases. The first restriction endonuclease recognizes the site having more than six bases and separates at a single site within each member of the anchor primer mix. The second restriction endonuclease is an endonuclease that recognizes a sequence of four nucleotides. Such endonucleases typically separate multiple sites in most cDNAs. Typically, the first restriction endonuclease is Notl and the second restriction endonuclease is MspI • Notl enzyme does not separate within most cDNAs. This is desirable to minimize the loss of cloned inserts that could result from the separation of the cDNAs at positions other than the anchoring site.

Alternatively, the second restriction endonuclease can be Taql, Maell or HinPlI. The use of the above three restriction endonucleases can detect rare mRNAs that are not separated by MspI. The second restriction endonuclease generates a compatible 5 'overhang for cloning into the desired vector, as discussed below. This cloning, for the vector chosen from the group consisting of pBC SK *, pBS SK \ pBC SKVDGT1, pBS SK + / DGT2 and pBS SKVDGT3, is within the Clal site, as discussed below. Alternatively, the second restriction endonuclease may be Sau3AI. The use of this restriction endonuclease can also detect rare mRNAs that are not separated by MspI. The second restriction endonuclease generates a compatible 5 'overhang for cloning into the desired vector, as discussed below. This cloning for the pBC vector SKVDGT4 is within the BamHI site, as discussed below. Alternatively, the second restriction endonuclease can be NalIII. The use of this restriction endonuclease can also detect rare mRNAs that are not separated by MspI. The second restriction endonuclease generates a compatible 5 'overhang for cloning into the desired vector, as discussed in the following. This cloning for the vector pBS SK + / DGT5, is in the Sphl site, as discussed in the following.

Alternatively, other suitable restriction endonucleases can be used to detect cDNAs that are not separated by the above restriction endonucleases. The second suitable restriction endonucleases that recognize a sequence of four nucleotides are Mbol, Dpnll, Sau3AI, Tsp509I, HpalI, Bfal, Cspßl, MseI, Hhal, NlalII, Taql, MSPI, Maell and HinPlI. The first suitable restriction endonucleases recognizing more than six bases are AseI, Bael, Fsel, Notl, PacI, Pmel, PpuMI, RsrII, SapI, SexAI, Sfil, SqfI, Sg_fI, SqrAI, Srfl, Sse8387I and SwaI. Conditions for cDNA digestion are well known in the art and are described, for example, in J. Sambrook et al., "Molecular Cloning: A Laboratory Manual," Vol. 1, Chap. 5, "Enzymes Used in Molecular Cloning." C. Insertion of the separated cDNA into a vector The cDNA sample separated with the first and second restriction endonucleases is then inserted into a vector. In general, a suitable vector includes a multiple cloning site having a Notl restriction endonuclease site. A suitable vector is the pBC SK + plasmid which has been separated from the restriction endonucleases Clal and Notl. The vector contains a bacteriophage-specific promoter.

Typically, the promoter is a T3 promoter, an SP6 promoter, or a T7 promoter. A preferred promoter is a bacteriophage T3 promoter. The separated cDNA is inserted into the promoter in an orientation that is antisense to the bacteriophage-specific promoter (Figures 1 and 2). In another preferred embodiment, the separated cDNA is inserted into the promoter in an orientation that is direct with respect to the bacteriophage-specific promoter (Figure 8). In a preferred embodiment, the vector includes a multiple cloning site having a nucleotide sequence which is selected from the group consisting of SEQ. FROM IDENT. NO: 9, SEC. FROM IDENT. NO: 10, SEC "of ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. Preferred vectors are based on the plasmid vector pBC SK * or pBS SK * (Stratagene ), in which a portion of the nucleotide sequence at positions 656 to 764 has been removed and replaced with a sequence of at least 110 nucleotides including a restriction endonuclease site NotI.This region, referred to as the cloning site Multiple (MCS) encompasses the portion of the nucleotide sequence from the Sacl site to the Kpnl site A suitable plasmid vector, such as pBC SK * or pBS SK *, is digested with a suitable restriction endonuclease to remove at least 100 nucleotides of the multiple cloning site In the case of pBS SK *, the restriction endonucleases suitable for removing the multiple cloning site are Sacl and Kpnl A portion of cDNA comprising a new multiple cloning site, having ends that are compatible with Notl and C After digestion with the first and second restriction endonucleases, it is cloned into the vector to form a suitable plasmid vector. Preferred cDNA portions comprise new multiple cloning sites include those having the nucleotide sequences described in SEQ. FROM IDENT. NO: 9, SEC. FROM IDENT. NO: 10 and SEC. FROM IDENT. NO: 11. The cDNA clones are linearized by digestion with a single restriction endonuclease that recognizes a sequence having more than six bases including the four nucleotide sequence of the second restriction endonuclease site. A plasmid vector, referred to as pBC SK * / DGT1, comprises the MCS of SEC. FROM IDENT. NO: 9. The pairs for the second restriction endonuclease and the linearization restriction endonuclease (stage E in the following) are, respectively: MspI and Smal; HinPlI and NarI; Taql and Xhol; Maell and AatII. Another preferred plasmid vector, referred to herein as pBS SK * / DGT2, comprises the MCS of SEQ. FROM IDENT. NO: 10 and prepared as described above for pBC SK * / DGT1. The multiple cloning site does not accept cDNA inserts produced using Maell. Therefore, for pBS SK * / DGT2, the pairs of the second restriction endonuclease and the linearization restriction endonuclease (from stage E in the following), are respectively: MspI and Smal; HinPlI and NarI; and Taql and Xhol. Another preferred plasmid vector, referred to as pBS SK * / DGT3, comprises the MCS of SEC. FROM IDENT. NO: 11. The pairs of the second restriction endonuclease and the linearization restriction endonuclease (from step E in the following) are, respectively: MspI and Smal; HinPlI and NarI; and Taql and Xhol; Maell and AatII. Another preferred plasmid vector, referred to herein as pBC SK + / DGT, comprises the MCS of SEC. FROM IDENT. NO: 12. The pair of the second restriction endonuclease and the linearization restriction enzymes (from step E in the following) suitable for use with this vector are, respectively, Sau3AI and BqlII. Another preferred plasmid vector, referred to as pBS SK * / DGT5, comprises the MCS of SEC. FROM IDENT. NO: 13. A pair of second restriction endonuclease and linearization restriction endonuclease enzymes (from step E in the following) suitable for use with this vector are, respectively, NalII and Ncol. In a preferred embodiment, the vector includes a vector filler sequence comprising an internal vector filler restriction endonuclease site between the first and second vector restriction endonuclease sites. In one such embodiment, the linearization step includes digestion of the vector with a restriction endonuclease which separates the vector at an internal vector filler restriction endonuclease site. In another such embodiment, the restriction endonuclease used in the linearization step also separates the vector at an internal vector filler restriction endonuclease site.

D. Transformation of an appropriate host cell The vector into which the separated DNA is inserted is then used to transform a suitable host cell that can be efficiently transformed or transfected by the vector containing the insert. Suitable host cells for cloning are described, for example, in Sambrook et al., "Molecular Cloning: A Laboratory Manual," supra. Typically, the host cell is prokaryotic. A particularly suitable host cell is an E. coli strain. A suitable strain of E. coli is MC 1061. Preferably, a small aliquot is also used to transform E. coli strain XLl-Blue so that the percentage of clones with inserts is determined from the relative percentages of blue and white colonies. on X-gal plates. Only libraries that exceed 5 x 105 recombinants are typically acceptable.

E. Generation of linearized fragments The plasmid preparations are then made from each of the cDNA libraries. The linearized fragments are then generated by digestion with at least one restriction endonuclease. In one embodiment, the vector is the pBC SK * plasmid and uses MspI as the second restriction endonuclease and as the linearization restriction endonuclease. In another embodiment, the vector is the plasmid pBC SK *, the second restriction endonuclease is chosen from the group consisting of MspI, Maell, Taql and HinPlI and the linearization is carried out by a first Smal digestion, followed by a Second digestion with a mixture of Kpnl and Apal. In other embodiments, the vector is selected from the group consisting of pBC SK * / DGT1, pBS SK * / DGT2, pBS SK * / DGT3, pBC SK * / DGT4 and pBS SK * / DGT5. In such embodiments, a suitable enzyme combination is provided wherein the second restriction endonuclease is MspI and the restriction endonuclease used in the linearization step is Smal. Another suitable combination is provided wherein the second restriction endonuclease is Taql and the restriction endonuclease used in the linearization step is XhoI. An additional suitable combination is provided where the second restriction endonuclease is HinPlI and the restriction endonuclease used in the linearization step is Narl. Another suitable additional combination is provided where the second restriction endonuclease is Maell and the restriction endonuclease used in the linearization step is AatII. If the vector is pBC SK + / DGT4, another suitable combination is provided by Sau3AI as the second restriction endonuclease and BglII with the restriction endonuclease used in the linearization step. If the vector is pBC SK * / DGT5, another suitable combination is provided by NlalII as the second restriction endonuclease and Ncol as the restriction endonuclease used in the linearization step. In general, in the linearization step, described in detail in section F below, any plasmid vector lacking a cDNA insert is separated at the 6 nucleotide recognition site (underlined in Figure 7A) for Smal, NarI, Xhol or AatII is located between the Notl site and the Clal site, and the recognition site that has more than six bases for the Smal, NarI, Xhol or Aat11 sites that is 3 'from the Clal site. In contrast, plasmid vectors containing inserts can be separated at the 6 nucleotide recognition site for the Smal, NarI, Xhol or Aat11 sites that are 3 'to the Clal site.

F. Generation of cRNA The next step is a generation of a cRNA preparation of antisense cRNA transcripts. This is done by incubating the linearized fragments with an RNA polymerase capable of initiating transcription from the bacteriophage-specific promoter. Typically, as discussed in the above, the promoter is a T3 promoter and the polymerase is therefore T3 RNA polymerase. The polymerase is incubated with the linearized fragments and the four ribonucleoside triphosphates under conditions suitable for synthesis (Ambion, Austin, TX).

G. Transcription of the cDNA of the first chain The first strand cDNA is transcribed using Moloney murine leukemia virus reverse transcriptase (MMLV) (Life Technologies, Gaithersburg, MD). With this reverse transcriptase, the annealing is carried out at 42 ° C and the transcription reaction at 42 ° C. The reaction uses a primer which is 15 to 30 nucleotides in length and is complementary to the 5 'flanking vector sequence.

In another embodiment, the cRNA is transcribed using a thermostable reverse transcriptase and a primer as described in the following. A preferred transcriptase is avian recombinant reverse transcriptase, known as ThermoScript RT, available from Life Technologies (Gaithersburg, MD). This promotes high fidelity complementarity between the primer and the cRNA. The primer used is at least 15 nucleotides in length, which corresponds in sequence to the 3 'end of the bacteriophage-specific promoter. Another suitable transcriptase is the recombinant reverse transcriptase of Thermus thermophilus, known as rTth, available from Perkin-Elmer (Nwalk, CT). When the bacteriophage-specific promoter is the T3 promoter, the primers typically have the sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G (SEQ ID NO: 14) or G-A-G-C-T-C-C-C-C-G-C-G-G-T (SEQ ID NO: 47).

H. Generation of the first PCR product The next step is the use of the transcription product as a template for a polymerase chain reaction with a first set of primers as described in the following, to produce fragments amplified by polymerase chain reaction.

In general, the product of the first strand cDNA transcriptase is used as a template for a polymerase chain reaction with a first 3 'PCR primer and a first 5' PCR primer to produce fragments amplified by polymerase chain reaction. The first 3 'PCR primer is typically 15 to 30 nucleotides in length, and is complementary to the sequences of the 3' flanking vector between the first restriction endonuclease site and the site that defines the initiation of transcription by the promoter-specific promoter. bacteriophage The first 5 'PCR primers have a 3' terminal part consisting of -NL where "-L" is one of the four deoxyrrinucleotides A, C, G or T, the primer being 15 to 30 nucleotides in length complementary to the 5 'flanking vector sequence with the primer complementarity extending into the nucleotide of the insert specific nucleotides of the cRNA, wherein a different one of the first 5' PCR primers is used in each of the four different subacumulates. When the pBC SK + plasmid separated with Clal and Notl is selected, a suitable 3 'PCR primer is selected from the group consisting of GAGCTCCACCGCGGT (SEQ ID NO: 47) and GAGCTCGTTTTCCCAG (SEQ ID NO: 48) . When the bacteriophage-specific promoter is the T3 promoter, a suitable 5 'PCR primer can have the sequence GGTCGACGGTATCGGN (SEQ ID NO: 22) where N is either A, G, C or T. Typically, PCR is performed using a PCR program of 15 seconds at 94 ° C for denaturation, 15 seconds at 50 ° C - 65 ° C for annealing or annealing and 30 seconds at 72 ° C for synthesis in a suitable thermal cycler such as PTC-200 ( MJ Research) or the Perkin-Elmer 9600 (Perkin-Elmer Cetus, Norwalk, CT). The annealing temperature is optimized for the specific nucleotide sequence of the primer, using principles well known in the art. The high temperature annealing step minimizes artifacts of a bad priming by the first 5 'PCR primer at its 3' end and promotes high fidelity copying.

I. Generation of the second PCR product The next step is the use of the products of the first PCR reaction as templates for a second polymerase chain reaction with a second set of primers as described in the following to produce a second set of fragments amplified by chain reaction of polymerase In general, the product of the first PCR reaction is used as a template for a polymerase chain reaction with a second 3 'PCR primer and a second 5' PCR primer to produce fragments amplified by polymerase chain reaction. The second 3 'PCR primer is typically 15 to 30 nucleotides in length and is complementary to the 3' flanking vector sequences between the first restriction endonuclease site and the site defining the initiation of transcription by the bacteriophage-specific promoter. . The second 5 'PCR primer is defined with a 3' terminal part consisting of Nx Nx, where Nx is identical to the N 'used in the first polymerase chain reaction for that subacumulate, "N" is as in step (H) and "x" is an integer from 1 to 5, the primer is 15 to 30 nucleotides in length and is complementary to the sequence of the 5 'flanking vector with the complementarity of the primer extending through the specific nucleotides of cRNA insert in a number of nucleotides equal to "x" + 1, where a different one of the second primers of 5 'PCR is used in subacumulates different from the second series of subacumulates and where there are 4X of subacumulates in the. second series of subacumulados for each of the subacumulados in the first set of subacumulados. In another embodiment, the primers used are: (a) a second 3 'PCR primer corresponding in sequence to a sequence in the vector adjacent to the insertion site of the cDNA sample in the vector; and (b) a 5 'PCR primer that is selected from the group consisting of: (i) the first 5' PCR primer which is used in the first PCR reaction for that subacumulate; (ii) the first 5 'PCR primer from which the cDNA of the first chain is made for that subacumulate extending in its terminal 3' part by an N-terminal residue; (iii) the first 5 'PCR primer used for that subacumulate extending in its 3' terminal part by two additional residues -NN, (iv) the first 5 'PCR primer used for that subacumulate extending in its 3' part terminal for three additional residues -NNN; and (v) the first 5 'PCR primer used for that subacumulate extending in its 3' terminal part by four additional residues -NNNN, wherein N may be any of A, C, G or T. The PCR primers Suitable 3 'are selected from the group consisting of GAGCTCCACCGCGT (SEQ ID NO: 47) and GAGCTCGTTTCCCCAG (SEQ ID NO: 48). When the promoter specific for bacteriophage is the T3 promoter, a suitable 5 'PCR primer is chosen from the group consisting of the sequences: A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO: 16); A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N (SEQ ID NO: 17); A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ.

IDENT. NO: 18); G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 22) G_T_C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO: 23) T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N (SEQ ID NO: 24) C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 25! G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N (SEQ ID NO: 26) A-C-G-G-T-A-T-C-G-G-N-N-N-N-N-N (SEQ ID NO: 16); A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N (SEQ ID NO: 19); and A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N-N (SEQ ID NO: 20). Typically, PCR is performed using a PCR program of 15 seconds at 94 ° C for denaturation, 15 seconds at 50 ° C - 65 ° C for annealing or annealing and 30 seconds at 72 ° C for synthesis in a suitable thermal cycler such as PTC -200 (MJ Research) or the Perkin-Elmer 9600 (Perkin-Elmer Cetus), Norwalk, CT). The annealing temperature is optimized for the specific nucleotide sequence of the primer, using principles well known in the art. The high temperature annealing step minimizes artifacts of a bad priming by the first 5 'PCR primer at its 3' end and promotes high fidelity copying. In the preferred embodiments, detection methods that do not use radioactive labels can also be used. For non-radioactive detection methods, one of the primers for the second PCR reaction is preferably conjugated to a fluorescent label. A suitable fluorescent label is selected from the group consisting of spiro (isobenzofuran-1 (3H), 9 '- (9H-xanthen) -3-one, 6-carboxylic acid, 3', 6'-dihydroxy-6-carboxyfluorescein ( 6-FAM, ABI); spiro (isobenzofuran-1 (3H), 9 '- (9H) -xanten) -3 -one, 5-carboxylic acid, 3', 6'-dihydroxy-5-carboxyfluorescein (5-FAM, Molecular Probes); spiro (isobenzofuran-1 (3H), 9'-xanten) -3-one, 3 ', 6'-dihydroxy-fluorescein (FAM, Molecular Probes); 9- (2,5-dicarboxyphenyl) -3,6-bis (dimethylamino) -xantylium (6-carboxymethylmethylrhodamine (6-TAMRA), Molecular Probes); 3, 6 -diamino- 9- (2 -carboxyphenyl) -xantilium (Rhodamine GreenMR, Molecular Probes); Spiro acid [isobenzofuran-1 (3H), 9'-xanten] -6-carboxylic acid, 5'-dichloro-3 ', 6'-dihydroxy-2', 7'-dimethoxy-3-oxo- (JOE, Molecular Probes ); inner salt of (2, 4 -disulf of enyl) -2,3, 6,7,12,13,16,17-octahydro 1H, 5H, 11H, 15H-xanthene [2,3,4-ij: 5, 6,7-i 'j'] diquinolizin-8-io, (Texas Red, Molecular Probes); 6- ((4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-propionyl) amino) hexanoic acid (BODIPY FL-X, Molecular Probes); 6- ((4,4-difluoro-l, 3-dimethyl -5- (4-methoxy phenyl) -4 -bora-3a, 4a-diaza-s-indacen-3-propionyl) amino) hexanoic acid (BODIPY TMR-X, Molecular Probes); 6- (((4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indacen-3-yl) phenoxy) acetyl) amino) -hexanoic acid (BODIPY TR - X, Molecular Probes); 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacen-3-pentanoic acid (BODIPY FL-C5, Molecular Probes); 4, 4-difluoro-5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-propanoic acid (BODIPY FL, Molecular Probes); 4, 4-difluoro-5-phenyl-4-bora-3a, 4a-diaza-s-indacen-3-propionic acid (BODIPY 581/591, Molecular Probes); 4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indacen-3-propionic acid (BODIPY 564/570, Molecular Probes); 4, 4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indacen-3-propionic acid; 6- (((4, 4-difluoro-5- (2-thienyl) -4 -bora-3 a, 4 a-diaza-s-indacen-3-yl) styryloxy) acetyl) aminohexanoic acid (BODIPY 630/650 , Molecular Probes); 6- (((4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indacen-3-yl) styryloxy) acetyl) aminohexanoic acid (BODIPY 650/665, Molecular Probes); and inner salt of 9- (2,4 (or 2,5) -dicarboxyphenyl) -3,6-bis (dimethylamino) -xantilium (TAMRA, Molecular Probes). Other suitable fluorescent labels include 4, 7, 2 ',', 5 ', 7'-hexachloro-6-carboxyfluorescein ("HEX", ABI), "NED" (ABI) and 4, 7, 2', 7 'tetrachlor 6 -carboxyfluorescein ("TET", ABI) which are known in the art. A preferred fluorescent label spiro (isobenzofuran-1 (3H), 9 '- (9H) -xanten) -3 -one, 6-carboxylic acid, 3', 6'-dihydroxy-6-carboxyfluorescein (6-FAM). In alternative modalities, autoradiographic detection methods can be used. In one embodiment, PCR is performed in the presence of 35S-dATP. Alternatively, PCR amplification can be carried out in the presence of radionuclide-labeled deoxyribonucleoside triphosphate, such as [32 P] dCTP or [33P] dCTP. However, autoradiographic detection is generally preferred for use of a deoxyribonucleoside triphosphate labeled with 3SS for maximum resolution. In a relative embodiment, the detection method uses oligonucleotides that are labeled with magnetic particles that are used and detected as described in U.S. Patent No. 5,656,429, the teachings of which are incorporated herein by reference. In one embodiment, the 3 nucleotides at the 3 'end of the first or second 5' PCR primers are linked by phosphorothioate linkages. See Mullins, J. I., de Noronha, C.M. Amplimers with 3 '-terminal phosphorothioate linkages resist degradation by vent polymerase and reduce Taq polymerase mispriming. PCR Methods Appl 1992 2 (2): 131-136; Ott, J. and Eckstein, F. Protection of oligonucleotide primers against degradation by DNA polymerase I. Biochemistry 1987 26 (25): 8237-8241; Uhlmann, E., Ryte, A., and Peyman, A. Studies on the mechanism of stabilization of partially phosphorothioated oligonucleotides against nucleolytic degradation. Antisense Nucleic Acid Drug Dev. 1997 7 (4): 345-350; Schreiber, G., Koch, E.M., and Neubert, W. J. Selective protection of in vitro synthesized cDNA against nucleases by incorporation of phosphorot hioa te - analogues. Nucleic Acids Res. 1985 13 (21): 7663-7672.

J. Electrophoresis The fragments amplified by polymerase chain reaction are then separated by a separation method such as electrophoresis to display bands representing the 3 'ends of the mRNAs present in the sample. Electrophoretic techniques for separating fragments amplified by PCR are well understood in the art and do not need to be mentioned in detail here. The corresponding PCR products are separated on denaturing DNA sequencing gels and visualized by laser-induced fluorescence. Alternatively, the corresponding PCR products are separated using capillary electrophoresis and visualized by laser-induced fluorescence. In a preferred embodiment, one of the primers for the second PCR reaction is conjugated to a fluorescent label. A suitable fluorescent label is selected from the group consisting of: spiro (isobenzofuran-1 (3H), 9 '- (9H-xanten) -3 -one, 6-carboxylic acid, 3', 6'-dihydroxy-6 -carboxyfluorescein (6-FAM, ABI); spiro (isobenzofuran-1 (3H), 9 '- (9H) -xanten) -3 -one, 5-carboxylic acid, 3', 6'-dihydroxy-5-carboxyfluorescein (5-FAM, Molecular Probes); spiro (isobenzofuran-1 (3H), 9'-xanten) -3-one, 3 ', 6'-dihydroxy-fluorescein (FAM, Molecular Probes); 9- (2,5-dicarboxyphenyl) -3,6-bis (dimethylamino) -xantylium (6-carboxytetramethylrhodamine (6-TAMRA), Molecular Probes); 3, 6-diamino-9- (2-carboxy nil) -xantilium (Rhodamine Green1 *, Molecular Probes); Spiro acid [isobenzofuran-1 (3H), 9'-xanten] -6-carboxylic acid, 5'-dichloro-3 ', 6'-dihydroxy-2', 7'-dimethoxy-3-oxo- (JOE, Molecular Probes); inner salt of (2, 4-di sul f of eni l) - 2, 3, 6, 7, 12, 13, 16, 17-octahydro 1H, 5H, 11H, 15H-xanthene [2,3,4-ij : 5, 6, 7-i 'j'] diquinolizin-8-io, (Texas Red, Molecular Probes); '6- ((4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-propionyl) amino) hexanoic acid (BODIPY FL-X, Molecular Probes); 6- ((4,4-difluoro-l, 3-dimethyl -5- (4-methoxy-enyl) -4-bora-3a, 4a-diaza-s-indacen-3-propionyl) -amino) -hexanoic acid (BODIPY TMR-X, Molecular Probes); 6- (((4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indacen-3-yl) phenoxy) acetyl) amino) -hexanoic acid (BODIPY TR - X, Molecular Probes); 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacen-3-pentanoic acid (BODIPY FL-C5, Molecular Probes); 4, 4-difluoro-5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-propanoic acid (BODIPY FL, Molecular Probes); 4, -difluoro-5-phenyl-4-bora-3a, 4a-diaza-s-índacen-3-propionic acid (BODIPY 581/591, Molecular Probes); 4, 4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indacen-3-propionic acid (BODIPY 564/570, Molecular Probes); 4, 4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indacen-3-propionic acid; 6- (((4,4-difluoro-5- (2-thienyl) -4-bora-3 a, 4 a-diaza-s-indacen-3-yl) styryloxy) acetyl) aminohexanoic acid (BODIPY 630 / 650, Molecular Probes); 6- (((4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, a-diaza-s-indacen-3-yl) styryloxy) acetyl) aminohexanoic acid (BODIPY 650/665, Molecular Probes); and inner salt of 9- (2,4 (or 2,5) -dicarboxyphenyl) -3,6-bis (dimethylamino) -xantilium (TAMRA, Molecular Probes). Other suitable fluorescent labels include 4, 7, 2 ', 4', 5 ', 7'-hexachloro-6-carboxyfluorescein ("HEX", ABI), "NED" (ABI) and 4, 7, 2', 7 'tetrachlor 6 -carboxyfluorescein ("TET", ABI) which are known in the art. Typically, fluorescence is used to detect the separated cDNA species. However, other detection methods can also be used, such as phosphorus imaging or autoradiography or magnetic detection. According to the scheme, cDNA libraries produced from each of the mRNA samples contain copies of the 3 'ends from the most distal site for MspI at the beginning of the poly (A) tail of all poly mRNAs (A) * in the starting RNA sample approximately according to the initial relative concentrations of the mRNA. Because both ends of the inserts for each species are defined exactly by sequence, their lengths are uniform for each species which allows their later visualization as separate rods in a gel, regardless of the tissue source of the mRNA. Typically, the intensity of the products exhibited after the electrophoresis is approximately proportional to the abundances of the mRNAs corresponding to the products in the original mixture. Typically, the method further comprises a step to determine the relative abundance of each mRNA in the original mixture from the intensity of the product corresponding to that mRNA after electrophoresis.

II. APPLICATIONS OF THE METHOD FOR EXHIBITION OF MRI PATTERNS The method described above for the detection of patterns of mRNA expression in a tissue and separation of these patterns by gel electrophoresis has many applications. One of these applications is its use for the detection of a field "in the pattern of expression of mRNA in a tissue associated with a physiological or pathological change." In general, this method comprises: (1) obtaining a first sample of a tissue that does not undergo physiological or pathological change; (2) determine the pattern of mRNA expression in the first tissue sample by performing the sequence-specific simultaneous identification method of the mRNAs that correspond to the members of an accumulated antisense cRNA that represent the 3 'ends of a population of the mRNAs as described above to generate a first display of bands representing the 3' ends of the mRNAs present in the first sample; (3) obtain a second sample of the tissue that has undergone the physiological or pathological change; (4) determining the mRNA expression pattern in the second tissue sample by performing the sequence-specific simultaneous identification method of the mRNAs corresponding to the accumulated antisense cRNA members representing the 3 'ends of a population of the MRNA as described above to generate a second display of the bands representing the 3 'ends of the mRNAs present in the second sample; and (5) comparing the first and second exhibits to determine the effect of the physiological or pathological change in the pattern of mRNA expression in the tissue.

Typically, the comparison is made in adjacent lanes of a single gel. Typically, a database comprising the data produced by the quantification of the display of the specific sequence products is constructed and maintained using suitable computer hardware and computer software. Preferably, such a database further comprises data regarding sequence relationships, gene mapping and cell distributions. In preferred embodiments, the length of at least part of the nucleotide sequence of the PCR products is compared to the expected values determined from a nucleotide sequence database. The tissue can be derived from the central nervous system. In particular, it can be derived from a structure within the central nervous system that is the retina, the cerebral cortex, the olfactory bulb, the thalamus, hypothalamus, anterior pituitary, posterior pituitary, hippocampus, nucleus accumbens, amygdala, striatum, cerebellum, stem cerebral, suprachyosmatic nucleus or spinal cord. When the tissue is derived from the central nervous system, the physiological or pathological change can be any of Alzheimer's diseases, parkinsonism, ischemia, addition to alcohol, addition to drugs, schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, depression and manic disorder. Depressive bipolar.

Alternatively, the method of the present invention can be used to study circadian variation, aging or long-term potentiation, the latter affecting the hypothalamus. Additionally, particularly with reference to the mRNA species that occur in particular structures within the central nervous system, the method can be used to study regions of the brain that are known to be involved in complex behaviors such as learning and memory, emotion, adhesion to drugs, neurotoxicity to glutamate, eating behavior, smell, viral infection, vision and movement disorders. This method can also be used to study the results of administering drugs or toxins to an individual by comparing the mRNA pattern of a tissue before and after administration of the drug or toxin. The results of electroshock therapy can also be studied. Alternatively, the tissue may be from an organ or organ system that includes the cardiovascular system, the pulmonary system, the digestive system, the peripheral nervous system, the liver, the kidney, skeletal muscle and the reproductive system, or any another organ or systems of body organs. For example, mRNA patterns of the liver, heart, kidney or skeletal muscle can be studied. Additionally, for any tissue, samples may be taken at various times so that a circadian effect on mRNA expression is discovered. Therefore, this method can ascribe particular mRNA species and their relationship in particular patterns of functioning or malfunction. Preferably, normal or neoplastic tissue comprises cells taken or derived from an organ or system of organs that are selected from the group consisting of the cardiovascular system, the lymphatic system, the respiratory system, the digestive system, the peripheral nervous system, the nervous system central, the enteric nervous system, the endocrine system, the integument (which includes skin, hair and nails), the skeletal system, (which includes bone and muscle), the urinary system and the reproductive system. In preferred embodiments, normal or neoplastic tissue comprises cells that are taken or derived from the group consisting of epithelium, endothelium, mucosa, glands, blood, lymph, connective tissue, cartilage, bone, smooth muscle, skeletal muscle, cardiac muscle , neurons, glial cells, spleen, thymus, pituitary, thyroid, parathyroid, adrenal cortex, suprarenal medulla, adrenal cortex, pineal, skin, hair, nails, teeth, liver, pancreas, lung, kidney, bladder, ureter, breast, ovary , uterus, vagina, testicles, prostate, penis, eyes and ears.

Similarly, the mRNA resolution method of the present invention can be used as part of an examination method for a side effect of a medicament. In general, such a method comprises: (1) obtaining a first tissue sample from an organism treated with a compound of known physiological function; (2) determine the pattern of mRNA expression in the first tissue sample by performing the sequence-specific simultaneous identification method of the mRNAs corresponding to the members of an accumulated antisense cRNA representing the 3 'ends of a population of the mRNAs, as described above, to generate a first-display of bands representing the 3 'ends of mRNA present in the first sample; (3) obtain a second tissue sample from an organism treated with a drug to be examined to determine its side effect; (4) determine the pattern of mRNA expression in the second tissue sample by performing the sequence-specific simultaneous identification method of the mRNAs corresponding to the members of an accumulated antisense cRNA representing the other 3 'end of a population of the mRNAs as described above, to generate a second display of bands representing the 3 'ends of the mRNAs present in the second sample; and (5) comparing the first and second display in order to detect the presence of mRNA species whose expression is not altered by the known compound, but which is affected by the drug to be examined, thus indicating a difference in the action of the drug to be examined and the known compound and therefore a side effect. In particular, this method can be used for drugs that affect the central nervous system, such as antidepressants, neuroleptics, tranquilizers, anticonvulsants, monoamine oxidase inhibitors and stimulants. However, this method can in fact be used for any drug that can alter the expression of mRNA in a particular tissue. For example, the effect on mRNA expression of antiparkisonism agents, skeletal muscle relaxants, analgesics, local anesthetics, cholinergics, antispasmodics, steroids, non-steroidal anti-inflammatory drugs, antiviral agents or any other drug capable of altering mRNA expression is they can study, and the effect is determined in a particular tissue or structure. An additional application of the method of the present invention is in obtaining a sequence of the 3 'ends of the mRNA species that are exhibited. In general, a method for obtaining the sequence comprises: (1) eluting at least one cDNA corresponding to an mRNA from an electropherogram in which the bands representing the 3 'ends of the mRNAs present in the sample are displayed; (2) amplify the cDNA eluted in a polymerase chain reaction; (3) cloning the amplified cDNA in a plasmid; (4) reproducing the DNA corresponding to the cloned DNA of the plasmid; and (5) sequencing the cloned cDNA. The cDNA that has been extracted can be amplified with the primers previously used in the second PCR step. The cDNA can then be cloned in PCR II (Invitrogen Sam Diego, CA) by cloning TA and ligation in the vector. The DNA mini-preparations can then be produced by standard techniques from subclones and a denatured portion and divided into two aliquots for automated sequencing of the Sanger dideoxy chain termination method. A commercially available sequencer, such as an ABI sequencer, can be used for automated sequencing. This will allow the determination of complementary sequences for most of the studied cDNAs, in the 50-500 bp length range, through the entire length of the fragment. These partial sequences can then be used to scan nucleotide databases such as GenBank using suitable computer equipment to recognize identities and sequence similarities using comparison and analysis programs such as BLASTN and BLASTX. Because this method generates sequences that only the 3 'ends of the mRNAs, it is expected that open reading frames (ORF) may be found only occasionally. For example, the 3 'untranslated regions of brain mRNAs are on average larger than 1300 nucleotides (J. G. Sutcliffe, 1988, supra). Potential ORFs can be screened for signatures of protein motifs. The cDNA sequences obtained can then be used to design primer pairs for semiquantitative PCR to confirm patterns of tissue expression. The selected products can also be used to isolate full-length cDNA clones for further analysis. The primer pairs can be used for SSCP-PCR (single-strand conformation polymorphism-PCR) genomic DNA amplification. For example, such amplification can be carried out from a panel of interspecific backcrossed mice to determine the binding of each PCR product to markers bound in advance. This can result in the mapping of new genes and can serve as a resource to identify mutant loci candidates from mapped mice and homologous human disease genes. SSCP-PCR uses synthetic oligonucleotide primers that amplify, by PCR, a small segment (100-200 bp) (M. Orita et al., "Detection of Polymorphisms of Human DNA by Gel Electrophoresis as Single-Strand Conformation Polymorphisms," Proc. Nati, Acad. Sci. USA 86: 2766-2770 (1989); M. Orita et al., "Rapid and Sensitive Detection of Point Mutations in DNA Polymorphisms Using the Polymerase Chain Reaction," Genomics 5: 874-879 (1989)). Cut cDNA fragments can be radiolabelled by techniques well known in the art for use in Northern blotting or for in situ hybridization to verify mRNA distribution and to learn the size and prevalence of mRNA length. full corresponding. The probe can also be used to examine a cDNA library to isolate clones for a more reliable and complete determination of the sequence. The labeled probes can also be used for any other purpose, for example for the study of in vitro expression.

III MIXTURES OF DEGENERATED PANELS OF PRIMERS Another aspect of the present invention is the primer panels and degenerate mixtures of primers suitable for the practice of the present invention. These include: (1) a panel of primers comprising 16 primers of the sequences A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ.

I DENT. NO: 16), wherein one or four deoxyribonucleotides A, C, G or T; (2) a panel of primers comprising 64 primers of the sequences AGGTCGACGGTATCGGNNN (SEQ ID NO: 17), (3) a panel of primers comprising 256 primers of the sequences AGGTCGACGGTATCGGNNNN (SEQ ID NO: 18) ); (4) a panel of primers comprising 1024 primers of the sequences A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N (SEQ ID NO: 19); (5) a panel of primers comprising 4096 primers of the sequences A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-N-N-N-N-N (SEQ ID NO: 20); (6) a panel of primers comprising 3 primers of the sequences A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 3); (7) a panel of primers comprising 12 primers of the sequences AACTGGAAGAATTCGCGGCCG-CAGGAATTTTTTTTTTTT-TT-TTTTVN (SEQ ID NO: 4), wherein V is a deoxyribonucleotide selected from the group consisting of A, C, and- G; (8) a panel of primers comprising 48 primers of the sequences A-A-C-T-G-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 5); (9) a panel of primers comprising 3 primers of the sequence G-A-T-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 6); (10) a panel of primers comprising 12 primers of the sequence G-A-T-T-T-C-A-A-C-T-G-G-A-G-C-G-G-C-C-C-C-G-C-A-G-G-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 7); (11) a panel of primers comprising 48 primers of the sequence G-A-T-T-T-C-A-A-C-T-G-G-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 8); (12) a panel "of primers comprising 4 different oligonucleotides, each having the sequence GGTCGACGGTATCGGN (SEQ ID NO: 22); (13) a panel of primers comprising 16 different oligonucleotides, each having the sequence GTCGACGGTATCGGNN (SEQ ID NO: 23); (14) a panel of chemists that comprises 64 different oligonucleotides, each having the sequence T-C-G-A-C-G-G-T-A-T-C-G-N-N-N (SEQ ID NO: 24); (15) a panel of primers comprising 256 different oligonucleotides, each having the sequence C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 25); (16) a panel of primers comprising 1024 different oligonucleotides, each having the sequence G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N (SEQ ID NO: 26); (17) a panel of primers comprising 4096 different oligonucleotides, each having the sequence A-C-G-T-A-T-C-G-G-N-N-N-N-N (SEQ ID NO: 27); (18) a degenerate mixture of primers comprising a mixture of primers 3 of the sequence AACTGGAAGAATTCGCGGCCG-CAGGAAT-TT-TTTTTTTTTTTTTTTV (SEQ ID NO: 2), each of the 3 primers is present in approximately an equimolar amount ( 19) a degenerate mixture of primers comprising a mixture of primers 3 of the sequence A-A-CTGGAAGAATTCGCGGCCGCA-GGAAT-TT-TTTTTTTTTTTTTTTVN (SEQ ID NO: 4), each of the 12 primers is present in approximately one equimolar amount (20) a degenerate mixture of primers comprising a mixture of primers 48 of the sequence AACTGGAAGAATTCGCGGCCG-CAGGAA-TT-TTTTTTTTTTTTTTTTVNN (SEQ ID NO: 5), each of the 48 primers is present in approximately one amount equimolar (21) a degenerate mixture of primers comprising a mixture of primers 3 of the sequence G-A-A-T-T-C-A-C-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T (SEQ. FROM IDENT. NO: 6), each of the 3 primers is present in approximately an equimolar amount; (22) a degenerate mixture of primers comprising a mixture of primers 12 of the sequence G-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-C-C-C-C-G-C-A-G-G-A-A-TT-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 7), each of the 12 primers is present in approximately an equimolar amount; and (23) a degenerate mixture of primers comprising a mixture of primers 48 of the sequence GAATTCAACTGGAAGCGGCCC-GCAGGAA-TT-TTTTTTTTTTTTTTTTVNN (SEQ ID NO: 8), each of the 48 primers is present in approximately an equimolar amount .

IV. SPECIFIC EXAMPLES OF PREFERRED MODALITIES Example 1: Application of the Improved Method The improved method of the present invention is based on the observation that virtually all eukaryotic mRNAs conclude with a poly (A) tail, but, unlike the differential display (Liang, P. and AB Pardee (1992) Differential display of eukaryotic messenger RNA by means of a polymerase chain reaction, Science 257: 967-971), the method of the present invention uses the specificity of the primer bound to the tail only to bind a site on each mRNA, and not to subdivide the mRNAs into accumulated The improved method is illustrated in three embodiments in Figures 1, 2 and 8. In general, the double-stranded cDNAs generated from cytoplasmic RNA enriched with poly (A) extracted from the tissue samples of interest using an equimolar mixture of all of the 48 5'-biotinylated anchor primers of a set to initiate reverse transcription (Figures 2 and 8) (Gubler, U. and B. Hoffman (1983) A simple and very efficient method for generating cDNA libraries. 25: 263-269 (Schibler, K., M. Tosi, A.C. Pittet, L. Fabiani and P.K. Wellauer (1980) Tissue-specific expression of mouse amylase genes, J. Mol. Biol. 142: 93-116). One such suitable assembly is AACTGGAAGAATTCGCGGCCG-CAGGAATTTTTTTTTTTTTTT-TTTVNN (SEQ ID NO: 5) wherein V is A, C or G and N is A, C, G or T. A member of this mixture of 48 primers of Anchor initiates synthesis at a fixed point at the 3 'end of all copies of each species of mRNA in the sample, thus defining a 3' endpoint for each species, resulting in double-stranded biotinylated cDNA. Each biotinylated double-stranded cDNA sample is cleaved with MpsI restriction endonuclease, which recognizes the CCGG sequence. The 3 'fragments of the cDNA are then isolated by capturing the biotinylated cDNA fragments on a substrate coated with streptavidin. Suitable substrates coated with idina strep include microtiter plates, PCR tubes, polystyrene spheres, paramagnetic polymer spheres and paramagnetic porous glass particles. A preferred substrate coated with streptavidin is a suspension of paramagnetic polymer spheres (Dynal, Inc., Lake Success, NY). After washing, the substrate coated with streptavidin and the captured biotinylated cDNA fragments, the product of the cDNA fragment is released by digestion with Notl, which separates a sequence of 8 nucleotides within the anchor primers but rarely within the derived from cDNA mRNA. The Mspl-Notl 3 'fragments, which are of uniform length for each species of mRNA, are directionally ligated in the plasmid separated by Clal, Notl, pCB SK + (Stratagene, La Jolla, CA) in an antisense orientation with respect to the promoter. T3 of the vector, and the product is used to transform Escherichia coli SURE cells (Stratagene). The ligation regenerates the Notl site but not the Mspl site. Each library contains an excess of 5 x 105 recombinants to ensure a high probability that the 3 'ends of all mRNAs with concentrations of 0.001% or greater are represented multiply. Plasmid preparations (Qiagen) are made from the DNA library of each sample under study. An aliquot of each library is digested with MspI, which carries out the linearization by separation at several sites within the parent vector while leaving the 3 'cDNA inserts and their flanking sequences, including the T3 promoter intact. The product is incubated with T3 polymeric RNAs (MEGAScript, Ambion) to generate antisense cRNA transcripts from the cloned inserts containing known vector sequences that make contact with the lPSp and NOR sites of the original cDNAs. This step prevents contamination of each cRNA sample to a different degree with plasmid transcripts without insert, which can lead to variability in the efficiency of subsequent PCRs for different samples due to the differential competence for primers. However, the polylinker region of the parental vector contains a site for MspI between its Clal and Notl sites and, therefore, the MspI digestion step removes the 5 'tag from the mRNA transcribed from plasmids without insert, which it renders them inert in the stages of product amplification described in the following. The plasmid DNA is removed from the mixture of antisense cRNA transcripts by incubation with DNase-free DNase. In this step, each of the cRNA preparations is processed in a three-step manner. In step 1, 250 ng of cRNA is converted to the cDNA of the first strand using the 5 'RT primer (5PRIMER (5 primer) in Figures 1 and 2 and 8) AGGTCGACGGTATCGG, (SEQ ID NO: 14) ). In step 2, 400 pg of cDNA product is used as a PCR template in four separate reactions with each of the four 5 'PCR primers of the form GGTCGACGGTATCGGN (SEQ ID NO: 22), each paired with a PCR primer 3 '"universal" 3' GAGCTCCACCGCGGT (SEQ ID NO: 47), using the program: 94 degrees Celsius, 15 seconds; 65 degrees Celsius, 15 seconds; 72 degrees Celsius, 60 seconds; 20 cycles.

In step 3, the product of each subacumulate is further divided into 64 subsubacumulates (2 ng in 20 μl) for the second PCR reaction, with 100 ng of each of the fluoresceinated 3 '"universal PCR primer, the oligonucleotide GAGCTCCACCGCGGT ( SEQ ID NO: 47) conjugated to 6-FAM and the appropriate 5 'PCR primer of the form CGACGGTATCGGNNNN (SEQ ID NO: 25) using the program: '94 degrees Celsius, 15 seconds; X degrees celsius, 15 seconds; 72 degrees Celsius, 30 seconds, 30 cycles including an annealing step at a temperature X slightly above Tm of each 5 'PCR primer to minimize a mismatch that may constitute an artifact and promote a high fidelity copy. Each step of polymerase chain reaction is performed in the presence of TaqStar antibody (Clonetech). The products of the final polymerase chain reaction stage for each of the tissue samples are separated into a se of denaturing DNA sequencing gels using the automated ABI Prizm 377 sequencer. Data is collected using the GeneScan software package (ABI) and normalized for amplitude and migration. The complete execution of this se of reactions generates 64 sub-cumulative products for each of the four accumulations established by the 5 'Nx PCR primers for a total of 256 sub-cumulative products for the entire 5' N4 PCR primer set. To summarize, in this modality of the improved method (figure 2), the reverse transcriptase is used to generate a cDNA pool from cRNA with a primer that is not 5 'RT primer recognition of the 5PRIMER form (5 primer) (SEQ ID NO: 14) , Taq DNA polymerase is used in PCR (20 cycles) to generate double-stranded cDNA subacumulates generated with the 5 'PCR primer 5PRIMERN1 (SEQ ID NO: 11) as 5'-PCR primer and 3' PCR primer ( ID SECTION NO: 47). The final PCR is car out for 30 cycles using '2 ng of DNA template and 100 ng of each primer 5 RIMER3N1N2N1N4 (SEQ ID NO: 25) and 3' PCR primer (SEQ ID NO: 47) ) conjugated to 6-FAM. Two human mRNA samples of human MG63 osteosarcoma cells lacking serum are analyzed (figure 3, panel A) and with serum added (figure 3, panel B). The data shown is generated with a 5 '-PCR primer (CGACGGTATCGGGGTG (SEQ ID NO: 42) paired with the 3' "universal" primer (SEQ ID NO: 47) labeled with 6-carboxyfluorescein (6FAM) , ABI) in the terminal 5 'part The PCR reaction products are separated by gel electrophoresis in 4.5% acylamide gels and fluorescence data acquired in ABI377 automated sequencers The data is analyzed using GeneScan software (Perkin-Elmer) In the three panels shown above, the relative abundance of the labeled PCR products (Y axis = relative fluorescence units) versus product length in base pairs is plotted.The high reproducibility of the method is shown in The lower part of the panel, which shows data from panels (A) and (B) superimposed using GeneScan software for comparisons of relative expression levels between samples.The largest application of the present invention is to compare mRNA expression profiles for two or more tissue samples. We compared the effect of the serum depletion / replenishment experiment on the panels of generated products. Most of the products in the pair of samples co-migrated and were of comparable amplitudes. Less than 10% of the products present amplitudes that differ by a factor of two or more, and these were distributed approximately equally between species induced by replenishment of serum and those repressed by replenishment. Many products, some of which are represented differentially in the two panels, seem to migrate in positions that coincide with the predicted DST based on the data extracted from GenBank., and therefore have candidate identities. To test these candidate identities, oligonucleotides corresponding to SP IMER ^ ^^ (SEQ ID NO: 25) were synthesized for each candidate extended at the 3 'end with 14 additional nucleotides from the sequences adjacent to the MspI sites. terminal in the GenBank sequences. These were paired with fluorescent 3PRIMER (SEQ ID NO: 47) in the PCRs using Nx cDNA as substrate.

EXAMPLE 2 Recognition Specificity in Modalities of the Method Using a PCR Stage and Two Stages of PCR: Analysis of PCR Products The advantages of the basic method modalities including two PCR steps are demonstrated using MG63 cells lacking serum and serum. For the two-step PCR variant of the basic method (Figure 1 and Figure 2), reverse transcriptase is used to generate a cDNA accumulation from cRNA with a primer and is not recognition (NO) of the form 5PRIMER (SEQ ID NO: 14); then Taq DNA polymerase is used in PCR (20 cycles) to generate subacumulates of double-stranded cDNA with 5PRIMERN1 (SEQ.

IDENT. NO: 22) as a 5 '-PCR primer and 3' PCR primer (SEQ ID NO: 47). In a modification of the PCR step, reverse transcriptase is used to generate four cDNA subacumulates from cRNA by transcription initiation with one of the four NI primers in the form of 5PRIMERN1 (SEQ ID NO: 22). In both methods, the final PCR is carried out for 30 cycles using 2 ng of a DNA template and 100 ng of each 5 'PCR primer (SEQ ID NO: 25) and a labeled 3' PCR primer with 6-FAM (SEQ ID NO: 47). The labeled PCR fragments are separated by electrophoresis in automated DNA sequencers (ABI377) and analyzed by Genescan software. The results are presented in Figure 4. The primer data 109T (CGACGGTATCGGTGCA, (SEQ ID NO: 43) and 45A (CGACGGTATCGGAGCA, (SEQ ID NO: 44), which differ only in position NI (with bold), are shown for samples lacking serum (Figures 4A, 4C, 4E and 4G) and with serum (Figures 4B, 4D, 4F and 4H). The PCR products generated with 109T and 45A appear to be almost identical for templates produced by the one-step PCR variant (compare Figure 4A with Figure 4C and Figure 4B with Figure 4D.) In contrast, products detected after PCR from templates produced using the two-step method PCR are generally very different (compare Figure 4E with Figure 4G and Figure 4F with Figure 4H.) The two-step PCR method of the method therefore provides a substantial improvement over the closest previously available method.

Example 3: Specificity of Recognition in the Modalities of Method Using One Step of PCR and Two Stages of PCR: Cloning and Sequence Data The method of the present invention is performed on MG63 cells lacking serum and treated with serum using either the one-step PCR (table I) or two-step PCR (table II). In the experiment shown in Table I, reverse transcriptase is used to generate four cDNA subacumulates from cRNA by initiation of transcription with one of the sets of four 5 'NI PCR primers (SEQ ID. NO: 22). For Table II, the reverse transcriptase is used to generate an accumulation of cDNA from cRNA with a RT 5 'non-recognition primer (SEQ ID NO: 14). Taq DNA polymerase is used in PCR (20 cycles) to generate double-stranded cDNA subacumulates with the 5 'PCR primer (SEQ ID NO: 22) and as a 3' PCR primer (SEQ ID NO. 47). The final PCR of both Table I and Table II is performed identically with a complete series of paired 5 'PCR 256 primers (SEQ ID NO: 25) with 3' PCR primer labeled with 6FAM (SEQ. DE IDENT NO: 47) using 2 ng of entry cDNA template. From the PCR reaction screens, differentially regulated molecules are identified and isolated for cloning and sequencing purposes. The DNA sequence data are obtained from individual clones and gene identification determined following database investigations using the BLAST algorithm. In the tables, the clones encounter are exact match of known human genes and are included with the. name of the gene and identification of the GenBank locus. The fidelity of the recognition step is determined using 5PRIMERN1 (SEQ ID NO: 22) either in reverse transcription fractions (table I) or PCR reactions (Table II) by tabulating the sequence match of the clone in the NI position with the GenBank sequence. In the two-step method, 5/22 clones match correctly in the NI position (essentially randomly) whereas in the three-step procedure, all the clones are found to correctly match the corresponding GenBank sequence data.

Table I SPECIFICITY OF RECOGNITION WITH A STAGE OF PCR NAME OF THE GEN IDENTIFICATION OF THE COINCIDENCE OF LOCUS D? GenBank THE NI POSITION Nma HSU23070 SI CDE1 binding protein HSCDEIBPA SI laminin receptor homolog S35960 SI protein C specific Ul snRNP HSU1RNPC SI Ubiquitin HSUBA52P SI MAD-3 HUMMAD3A NO a- tubulin HSTUBB2 NO Idl HSID1 NO NNMT HSNNMT2 NO BFGF HUMGFB NO SC35 HUMSC35A NO protein S14 ribosomal HUMRPS14 NO protein L30 ribosomal HUMRPL30A NO Na / K ATPase B3 HSU51 78 NO ribosomal protein L37A HSRPL37A NO IRF-2 HS1RF2 NO SRp20 HUMSRP20 NO Glioxalase II HSHAGH1 NO oncogene pim-1 HUMPIMl NO Endothelin-1 HUMEDN1B NO Metallothioneine II HUMMETIIPS NO homolog CRP3 S631S8 NO TABLE II: SPECIFICITY OF RECOGNITION WITH TWO STAGES OF PCR NAME OF THE GEN IDENTIFICATION OF THE COINCIDENCE OF LOCUS OF GenBank THE POSITION NOR MAD-3 HUMMAD3A YES Idl HSID1 YES Na / K ATPase B3 HSU51478 SI oncogene pim-1 HUMPIM1 YES endothelin-1 HUMEDN1B YES ribosomal protein S20 HUMRPS20 YES ribosomal protein SIO HUMRPS10 YES GADD45 HUMGADD45 YES AP-2 HSAP2 YES beta-2 microglobulin HUMB2M02 YES RDC-1 HSU67784 SI autoantigen 56K HUM56KAUTO SI NFKB1 HSNFX24 SI Protein-like protein Lon HSLONP SI protein that binds nucleotide U01833 SI insulinoma gene HUMIDB SI histone 2A.2 HUMH2A2A SI Note that five gene products highlighted in bold, MAD-3, Idl, Na / K ATPase B3, pim-1 oncogene and endothelin-1 are isolated in both experiments, and in each case the two-step PCR method produces a coincidence in the NL position, while the one-step PCR method does not. The two-step PCR method therefore provides a substantial improvement over the closest method available previously.

EXAMPLE 4 Improved Resolution That Is Obtained Using Biotinized Anchor Primers As indicated above, in a preferred embodiment, anchor primers are biotinized at their 5 'end (compare FIGS. 1 and 2). Biotinylated cDNA fragments can be captured using a substrate coated with streptavidin, preferably paramagnetic spheres coated with streptavidin (Dynal). Figure 5 compares the results of the standard basic method with those obtained during the anchor primers marked with magnetic spheres.

CDNA libraries are constructed using the standard technique (as indicated in figure 1) and the alternative modality of magnetic sphere (see figure 2) from aliquots of 2 μg of mRNA from five separate samples of mouse extract treated with haloperidol taken in a series in time (0, 0.75, 7 hours, 10 and 14 days). The results are shown in Figure 5 A-E (standard) and 5F-J (magnetic sphere). The results of primer PCR5 '170G (CGACGGTATCGGGGT, (SEQ ID NO: 45) and the 3' primer PCR labeled with 6-FAM (SEQ ID NO: 47) is shown in both cases for comparison. graph the relative abundance of the labeled PCR products (arbitrary fluorescence units on the Y axis) versus the length of the PCR product (base pairs) .The data from the magnetic sphere libraries (Figure 5F-J) show greater capacity of reproduction through the samples in a series over time (in fragment similarity as well as in the consistency of intensity values) and apparently less spurious short fragments (100-125 bp) in comparison with the data from a standard library technique (Figure 5A-E).

EXAMPLE 5: Demonstration of Linearity in the three-stage method Relationship of the height of the PCR product with the concentration of introduced cRNA To determine the range of linear amplification, a known addition is made in 4 independent accumulations of cRNA and processed by the method of the present invention as shown in Figure 2. The results are shown in Figure 6. The peak height (in units of relative fluorescence) corresponding to the synthetic RNA is measured and plotted versus the input concentration for the 4 samples. The data show averages of triplicate determinations; the error bars indicate the interval of _ + a standard error of the mean. A SalI -Notl cDNA fragment (SEQ ID NO: 51) is cloned into the library vector pBCSK +, linearized and produced by cRNA by transcription from the synthetic T3 promoter cRNA is constructed to provide an increase to a type of known size (492 bp) in PCR. Variable amounts of cRNA (0, 25, 100 or 250 pg) are introduced into a pool of 250 ng of cRNA before reverse transcription with primer N0 (SEQ ID NO: 14). 400 pg of cDNA is used as a template for the PCR reactions with the 5 'PCR primer (SEQ ID NO: 22) and the 3' PCR primers (SEQ ID NO: 47), respectively. An aliquot of 2 ng of cDNA in final PCR was used with 5 '221C PCR primer (C-G-A-C-G-T-A-T-C-G-G-C-T-C-A, SEQ ID NO: 46) and 3' PCR primer (SEQ ID NO: 47). The results shown in Figure 6 show that for a given tissue type, the peak height of the PCR product is proportional to the concentration of RNA introduced. The foregoing is intended to be illustrative of the present invention, but not limiting. Numerous variations and modifications of the present invention can be made without departing from the true spirit and scope of the invention.

LIST OF SEQUENCES < 110 > Hasel, Karl. Hilbush, Brian S. < 120 > Method to classify and determine the relative concentration of expressed messenger RNA < 130 > 98,429-A < 140 > < 141 > 1999-10-14 < 150 > US 09 / 186,869 < 151 > 04-11-1998 < 160 > 51 < 170 > Patentln Ver. 2.0 < 210 > 1 < 2ll 14 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 1 aactggaaga attc 14 < 210 > 2 < 211 > 14 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 2 gaattcaact ggaa 14 < 210 > 3 < 211 > 46 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 3 aactggaaga attcgcggcc gcaggaattt tttttttttt tttttv 46 < 210 > 4 < 211 > 47 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 4X > 0 > 4 aactggaaga attcgcggcc gcaggaattt tttttttttt tttttvn 47 < 210 > < 211 > 48 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 5 aactggaaga attcgcggcc gcaggaattt tttttttttt tttttvnn 48 < 210 > 6 < 211 > 47 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 4Q0 > 6 gaattcaact ggaagcggcc cgcaggaatt tttttttttt ttttttv 47 < 210 > 7 < 211 > 48 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 7 gaattcaact ggaagcggcc cgcaggaatt tttttttttt ttttttvn 48 < 210 > 8 < 211 > 49 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Synthetic description < 400 > 8 gaattcaact ggaagcggcc cgcaggaatt tttttttttt ttttttvnn 49 < 210 > 9 < 211 > 116 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of multiple cloning < 400 > 9 gagctccacc gcggtgtcac gactatctgc ggccgcatgc ccgggaatgg cgcctcgaga 60 cgtctttatc gataccgtcg acctcgaact cgagacgtcc cgggcgccta ggtacc 116 < 210 > 10 < 211 > 113 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: multiple cloning site < 400 > 10 gagctcgttt tcccagtcac gactatctgc ggccgcatgc ccgggaatgg cgcctcgaga 60 cgttatcgat tagcctgact gaagactcga gacgtcccgg gcgcctaggt acc 113 < 210 > 11 < 211 > 113 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: multiple cloning site < 400 > 11 gagctcgttt tcccagtcac gactatctgc ggccgcatgc ccgggaatgg cgcctcgaga 60 cgtctatatc gattagcctg actgaagact cgagacgtcc cgggctaggt acc 113 < 210 > 12 < 211 > 62 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: multiple cloning site < 400 > 12 gcggccgcat agatctgata tcggatcctc accacagagc tcagtgagag agatctctcg 60 ag 62 < 210 > 13 < 211 > 62 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: multiple cloning site < 400 > 13 gcggccgcat ccatgggata tcgcatgctc accacagtcg acagtgagag ccatggctcg 60 ag 62 < 210 > 14 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 14 aggtcgacgg tatcgg 16 < 210 > 15 < 211 > 17 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the synthetic primer artificial sequence < 400 > 15 aggtcgacgg tatcggn 17 < 210 > 16 < 211 > 18 < 212 > DNA < 13 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 16 aggtcgacgg tatcggnn 18 < 210 > 17 < r211 > 19 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 17 aggtcgacgg tatcggnnn 19 < 210 > 18 < 211 > 20 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 18 aggtcgacgg tatcggnnnn 20 < 210 > 19 < 211 > 21 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 19 aggtcgacgg tatcggnnnn n 21 < 210 > 20 < 211 > 22 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 20 aggtcgacgg tatcggnnnn nn 22 < 210 > 21 < 211 > 15 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > "Description of the artificial sequence: synthetic primer <400> 21 ggtcgacggt atcgg 15 < 210 > 22 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 22 ggtcgacggt atcggn 16 < 210 > 23 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 23 gtcgacggta tcggnn 16 < 210 > 24 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 24 tcgacggtat cggnnn 16 < 210 > 25 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 25 cgacggtatc ggnnnn 16 < 210 > 26 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 26 gacggtatcg gnnnnn 16 < 210 > 27 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 27 acggtatcgg nnnnnn 16 < 210 > 28 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 28 agctctgtgg tgaggatc < 210 > 29 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 29 gctctgtggt gaggatcn "18 <210> 30 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: synthetic primer < 213 > 30 < 211 > 18 < 212 > 400> 30 ctctgtggtg aggatcnn 18 < 210 > 31 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 31 tctgtggtga ggatcnnn 18 < 210 > 32 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > "Description of the Artificial Sequence: Synthetic Primer <400> 32 ctgtggtgag gatcnnnn 18 < 210 > 33 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 33 tgtggtgagg atcnnnnn 18 < 210 > 34 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 34 gtggtgagga tcnnnnnn 18 < 210 > 35 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 35 tcgactgtgg tgagcatg 18 < 210 > 36 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 36 cgactgtggt gagcatgn < 210 > , 37 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 37 gactgtggtg agcatgnn 18 < 210 > 38 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > The description of the artificial synthetic primer sequence < 400 > 38 actgtggtga gcatgnnn 18 < 210 > 39 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 39 ctgtggtgag catgnnnn 18 < 210 > 40 < 211 > 18 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 40 tgtggtgagc atgnnnnn < 210 > 41 < 211 > 18 < 2l2 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 41 gtggtgagca tgnnnnnn 18 < 210 > 42 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Artificial sequence description: synthetic primer < 400 > 42 cgacggtatc ggggtg 16 < 210 > 43 < 211 > 16 < 212 > DNA < 213 > "Artificial sequence <220> <223> Description of the artificial sequence synthetic primer <400> 43 cgacggtatc ggtgca 16 < 210 > , 44 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 44 cgacggtatc ggagca 16 < 210 > 45 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 45 cgacggtatc gggggt 16 < 210 > 46 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 46 dgacggtatc ggctca 16 < 210 > 47 < 211 > 15 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer * < 400 > 47 gagctccacc gcggt 15 < 210 > 48 < 211 > 16 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 48 gagctcgttt tcccag 16 < 210 > 49 < 211 > 22 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial synthetic primer sequence < 400 > 49 gtcttcagtc aggctaatcg gn 22 < 210 > 50 < 211 > 22 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 50 cctcgaggtc gacggtatcg gn 22 < 210 > 51 < 211 > 481 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial sequence: synthetic primer < 400 > 51 gtcgacggta tcggctcaag tgactgactg tctagaactt taccattacg gagagatgat 60 gatcagtaac caagattatc ttggactatc tttaggttct ttaaaaaaac tgcttattac 120 agctgaccta caacctttgt agatctttgt gcctgttatg taaaaagttt ggaatgtatt 180 gttaaactta gccaacgact ggcttttcag cagtgctcaa aagaagagta tcatcagctg 240 gagattttcc tgctatgctg tagcctacct ccccgatgtc ctttccgcta tatttggcaa 300 atgtattgat ttatggtctt ttgttctatg gctataagac tgcgtgtaaa cctctttcac 360 agtagaacat gtaattctgg gaaacccgaa tctctgttac taagcactat tcactcaaag 420 ttgcctcaga ataaactttc tttgggtttt aaaaaaaaaa aaaaaaaatt cctgcggccg 480 c 481 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (88)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An improved method for specific identification of simultaneous sequencing of mRNAs in a population of mRNA, characterized in that it comprises the steps of: (a) preparing a population of double-stranded cDNA from a population of mRNA using a mixture of anchor primers , each anchor primer has a 5 'terminal part and a 3' terminal part which includes: (i) a stretch of 7 to 40 T residues; (ii) a site for separation by a first restriction endonuclease that recognizes more than six bases, the site for separation is located towards the 5 'end portion relative to the stretch of the T residues; (iii) a first filler segment of 4 to 40 nucleotides, the first filler segment is located towards the 5 'end portion relative to the site for separation by the first restriction endonuclease; (iv) a second filler segment interposed between the site for separation by a first restriction endonuclease that recognizes more than six bases and the residue tract T, and (v) phase adjustment residues that are located at the 3 'end part of each of the anchor primers that are selected from the group consisting of -V, -VN, and -VNN, wherein V is a deoxyribonucleotide which is selected from the group consisting of A, C and G, and N is a deoxyribonucleotide which is selected from the group consisting of A, C, G and T, the mixture includes anchor primers which contain all possibilities for V and N; (b) separating the double-stranded cDNA population with the first restriction endonuclease and a second restriction endonuclease, the second restriction endonuclease recognizes a sequence of four nucleotides, to form a population of double-stranded cDNA molecules having first and second terminal parts, respectively; (c) inserting each double-stranded cDNA molecule of step (b) into a vector in an orientation that is antisense to a bacteriophage-specific promoter within the vector to form a population of constructs containing the inserted cDNA molecules , whereby 5 'and 3' flanking vector sequences are defined adjacent to the 5 'end portion of the direct strand of the inserted cDNA, and the 3' end portion of the direct strand, respectively, and the constructs have a sequence of 3 'flanking vector of at least 15 nucleotides in length between the first restriction endonuclease site and a site defining the start of transcription in the promoter; (d) transforming a host cell with the vector into which the separated cDNA has been inserted to produce vectors containing cloned inserts; (e) generating linearized fragments containing the cDNA molecules inserted by digestion of the constructs produced in step (c) with at least one restriction endonuclease that does not recognize sequences either inserted into the cDNA molecules or into the specific promoter of bacteriophage, but sequences are recognized in the vector, so that the resulting "linearized" fragments have a 5 'flanking vector sequence of at least 15 nucleotides within the 5' vector for the second terminal part of the chain cDNA molecules double; (f) generating a cRNA preparation of antisense cRNA transcripts by incubating the linearized fragments with a bacteriophage-specific RNA polymerase capable of initiating transcription from the bacteriophage-specific promoter; (g) generating first-strand cDNA upon transcription of the CRNA using a reverse transcriptase and a 5 'RT primer that is 15 to 30 nucleotides in length and that comp renders a nucleotide sequence that is complementary to the sequence of the 5 'flanking vector; (h) generate a first set of PCR sets by dividing the cDNA of the first strand in a first series of subacumulates and use the cDNA of the first strand as templates for a first polymerase chain reaction with a first 3 'PCR primer of 15 to 30 nucleotides in length which is complementary to the sequences of 3 'flanking vectors between the first restriction endonuclease site and the site defining the initiation of transcription by the bacteriophage-specific promoter and a first 5' PCR primer defined with a 3 'terminal part consisting of -N? / wherein "N" is one of the four deoxyribonucleotides A, C, G, or T, the first 5 'PCR primer is 15 to 30 nucleotides in length and complementary to the 5' flanking vector sequence with the first complementarity of 5 'PCR primers extending into a nucleotide of the specific nucleotides of the cRNA insert, wherein a different one of the first 5 'PCR primers are used in each of the four different subacumulates; (i) generate a second set of PCR products by further dividing the first set of PCR products in each of the first series of sub-cumulatives in a second set of subacumulates and using the first set of PCR products as templates for a second set polymerase chain reaction with a second 3 'PCR primer of 15 to 30 nucleotides in length that is complementary to the 3' flanking vector sequences between the first restriction endonuclease site and the site that defines the initiation of tcription by the promoter specific bacteriophage, and a second 5 'PCR primer defined with a 3' terminal part consisting of Nx Nx, where Nx is identical to the NL used in the first polymerase chain reaction for that subacumulate,, TN "is like in step (h) and "x" is an integer from 1 to 5, the primer is 15 to 30 nucleotides in length and complementary to the 5 'flanking vector sequence with the complementary primer age extending through the insert specific nucleotides of the cRNA at a number of nucleotides equal to "x" +1, wherein a different one of the second 5 'PCR primers are used in different subacumulates of the second series of subacumulates where there are 4X subacumulados in the second series of subacumulados for each of the subacumulados in the first set of subacumulados; and (j) separating the second set of PCR products to generate a display of sequence specific products representing the 3 'ends of the mRNAs present in the mRNA population. 2. The method according to claim 1, characterized in that the biotin portion is conjugated to the anchor primers. 3. The con fi dence method with claim 2, characterized in that the biotin portion is conjugated to the terminal 5 'part of the anchor primer. 4. The method according to claim 2, characterized in that the first restricted cDNA is separated from the rest of the cDNA in step b of claim 1 by contacting the first restricted cDNA with a substrate coated with streptavidin. The method according to claim 1, characterized in that the 3 nucleotides at the 3 t end of the first 5 'PCR primer are linked by phosphothioate linkages. 6. The method according to claim 1, characterized in that the 3 nucleotides at the 3 'end of the second 5' PCR primer are linked by phosphothioate linkages. 7. The con fi dence method with claim 1, characterized in that the 3 nucleotides at the 3 'end of the first and second 5' PCR primers are linked by phosphothioate linkages. 8. The method of confection with claim 1, characterized in that one of the primers for the second PCR reaction is conjugated to a fluorescent tag. 9. The method according to claim 8, characterized in that the fluorescent label is selected from the group consisting of: spiro (isobenzofuran-1 (3H), 9 '- (9H-xanthen) -3 -one, 6-carboxylic acid, 3', 6'-dihydroxy-6-carboxyfluorescein; spiro (isobenzofuran-1 (3H), 9 '- (9H) -xanten) -3-one, 5-carboxylic acid, 3', 6'-dihydroxy-5-carboxyfluorescein; spiro (isobenzofuran-1 (3H), 9'-xanten) -3-one, 3 ', 6 dihydroxy-fluorescein; 9- (2,5-dicarboxyphenyl) -3,6-bis (dimethylamino) -xantylium (6-carboxitetramethylrhodamine; 3, 6-diamino-9- (2-carboxyphenyl) -xantilium; Spiro acid [isobenzofuran-1 (3H), 9'-xanten] -6-carboxylic acid, 5'-dichloro-3 ', 6'-dihydroxy-2', 7'-dimethoxy-3-oxo; internal salt of (2, 4-di sul f of enyl) -2.3, 6, 7, 12, 13, 16, 17-octahydro 1H, 5H, 11H, 15H-xanthene [2, 3, 4 -ij: 5,6,7-i'j'3 diquinolizin-8-iom 6- ((4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-propionyl) amino) hexanoic acid; 6- ((4,4-difluoro-1,3-dimethyl-5- (4-methoxy phenyl) -4-bora-3a, 4a-diaza-s-indacen-3-propionyl) amino) hexanoic acid; 6- (((4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indacen-3-yl) phenoxy) acetyl) amino) -hexanoic acid; 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacen-3-pentanoic acid; 4, 4-difluoro-5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-propanoic acid; 4, 4 -dif luoro-5-f-enyl-4-bora-3a, 4a-diaza-s-indacen-3-propionic acid; 4, 4-difluoro-5- (4-f-enyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indacen-3-propionic acid; 4,4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indacen-3-propionic acid; 6- (((4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indacen-3-yl) styryloxy) acetyl) aminohexanoic acid; 6- (((4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indacen-3-yl) styryloxy) acetyl) aminohexanoic acid; Y inner salt of 9- (2,4 (or 2,5) -dicarboxyphenyl) -3,6-bis (dimethylamino) -xantylium; Y 4, 7, 2 ', 4', 5 ', 7'-hexachloro-6-carboxyfluorescein and 4,7,2', 7'-tetrachloro-6-carboxyfluorescein. * 10. The method according to claim 1, characterized in that the host cell is an Escherichia coli cell. 11. The method according to claim 1, characterized in that the phase adjustment residues in step (a) are -V-N-N. 12. The conformance method with claim 1, characterized in that the adjustment residues in step 8a) are -V-N. 13. The conformance method with claim 1, characterized in that the phase adjustment residues in step (a) are -V. 14. The conformance method with claim 1, characterized in that "x" in step (i) is 3. 15. The method of con fi dence with claim 1, characterized in that "x" in step (i) is 1. 16. The method according to claim 1, characterized in that the phase-adjusting residues in step (a) are -V-N-N and "x" in step (i) is 3. 17. The method according to claim 1, characterized in that the phase adjustment residues in step (a) are -V and the "x" in step (i) is 2. 18. The method according to claim 1, characterized in that the anchor primers each have 18 T residues in the waste section T. r 19. The method according to claim 1, characterized in that the first filler segment of the primers of Anchor has 14 residues in length. 20. The method according to claim 1, characterized in that the sequence of the first filler segment is G-A-A-T-T-C-A-A-C-T-G-G-A-A (SEQ ID NO: 2) 21, The method according to claim 1, characterized in that the bacteriophage-specific promoter is selected from the group consisting of the T3 promoter, the T7 promoter and the SP6 promoter. 22. The method according to claim 1, characterized in that the bacteriophage-specific promoter is the T3 promoter. 23. The method according to claim 1, characterized in that the primer for priming the cDNA transcription from cRNA has the sequence A-G-T-C-G-A-C-G-G-T-A-T-C-G-G (SEQ ID NO: 14). 24. The method according to claim 1, characterized in that the vector is the pBC SK + plasmid separated with Clal and Notl and the 3 'PCR primer in steps (h) and (i) is GAGCTCCACCGCGGT (SEQ ID NO. 47). 25. The method according to claim 1, characterized in that the vector is the pBC SK + plasmid separated with Clal and Notl and the 3 'PCR primer in steps (h) and (i) is GAGCTCGTTTTCCCAG (SEQ ID NO: 48). 26. The method according to claim 1, characterized in that the second restriction endonuclease recognizes a sequence of 4 nucleotides and is Mspl. 27. The method according to claim 1, characterized in that the second restriction endonuclease recognizing a sequence of four nucleotides is selected from the group consisting of Mbol, Dpnll, Sau3AI, Tsp509I, HpalI, Bfal, Csp.61, MseI, Hhal, NlalII, Taql, MspI, Maell, Sau3AI, BIIII and HinPII. 28. The method according to claim 1, characterized in that the first restriction endonuclease recognizing more than six bases is selected from the group consisting of AscI, Bael, Fsel, Notl, PacI, Pmel, PpuMI, RsrII, SapI, SexAI, Sfil , Sqfl, SgrAI, SrfI, SSe8387I and SwaI. 29. The method according to claim 1, characterized in that the first restriction endonuclease that recognizes more than six bases is Notl. 30. The method according to claim 1, characterized in that the restriction endonuclease used in step (e) has a nucleotide sequence recognition that includes the four nucleotide sequence of the second restriction endonuclease that is used in step (b) ). 31. The method according to claim 30, characterized in that the second restriction endonuclease is MSTOI and the restriction endonuclease used in step (e) is Smal. 32. The method of con? Rmity with claim 30, characterized in that the second restriction endonuclease is Taql and the restriction endonuclease used in step (e) is XhoI. 33. The method according to claim 30, characterized in that the second restriction endonuclease is HinPII and the restriction endonuclease used in step (e) is Narl. 34. The method according to claim 30, characterized in that the second restriction endonuclease is Maell and the restriction endonuclease used in step (e) is AatII. 35. The method according to claim 30, characterized in that the second restriction endonuclease is Sau3AI and the restriction endonuclease used in step (e) is BqlII. 36. The method according to claim 30, characterized in that the second restriction endonuclease is NlalII and the restriction endonuclease used in step (e) is Ncol. 37. A vector suitable for practicing the method according to claim 1, characterized in that the vector of step c) is in the form of a circular DNA molecule having a first and second vector restriction endonuclease sites flanking a sequence vector filler, and further comprising the step of digesting the vector with restriction endonucleases that separate the vector in the first and second vector restriction endonuclease sites. 38. The vector according to claim 37, characterized in that the vector filler sequence includes an endonuclease site of internal vector filler restriction between the first and second restriction endonuclease sites of the vector. 39. The vector of conmunity with claim 38, characterized in that step (e) includes digestion of the vector with a restriction endonuclease which separates the vector at the restriction endonuclease site of the internal vector filler. 40. A vector, characterized in that it is selected from the group consisting of the pBC SK + / DGT1 plasmids, pBS SK + / DGT2, pBS SK + / DGT3, pBC SK + / DGT4 and pBS SK + / DGT5. 4 * 1 A vector characterized in that it comprises a multiple cloning site which is selected from the group consisting of SEQ. FROM IDENT. NO: 9, SEC. FROM IDENT. NO: 10, SEC. FROM IDENT. NO: 11, SEC. FROM IDENT. NO: 12 and SEC. FROM IDENT. NO: 13 42. The method according to claim 1, characterized in that the mRNA population has been enriched from polyadenylated mRNA species. 43. The method according to claim 1, characterized in that the separation in step (j) of the amplified fragments is carried out by electrophoresis to display the products. 44. The method according to claim 43, characterized in that it further comprises the step (k) of determining the relative abundances of the fragments amplified by the peak heights of the products displayed separately. 45. The method according to claim 44, characterized in that step (k) of determining the relative abundances of the amplified fragments is carried out by measuring the fluorescence intensity of the fluorescently labeled products. * 46 The method according to claim 43, characterized in that the step of separating the fragments amplified by polymerase chain reaction by electrophoresis comprises the electrophoresis of the proteins in at least two gels. 47. The method according to claim 40, characterized in that it further comprises the steps of: (k) eluting at least one cDNA corresponding to an mRNA from an electropherogram in which bands representing the 3 'ends of the MRNA present in the sample; (1) amplifying the cDNA eluted in a polymerase chain reaction; (m) cloning the amplified cDNA in a plasmid; (n) producing DNA corresponding to the DNA cloned from the plasmid; and (o) sequencing the cloned cDNA. 48. A method for sequentially specific identification of mRNA sequences in a population of mRNA comprising the steps of: (a) isolating a population of mRNA; (b) preparing a population of double-stranded cDNA from the mRNA population using a mixture of anchor primers, each anchor primer having a 5'-terminal part and a 3'-terminal part and includes: (i) a stretch from 7 to 40 waste T; (ii) a site for separation by a first restriction endonuclease that recognizes eight bases, the site for separation is located towards the 5 'end portion in relation to the stretch of the T residues; (iii) a first filler segment of 4 to 40 nucleotides, the first filler segment is located towards the 5 'end portion relative to the site for separation by the first restriction endonuclease; (iv) a second filler segment interposed between the site for separation by the first restriction endonuclease that recognizes more than six bases, and the stretch of residues T, and (v) phase adjustment residues defined by one of -V, - VN, or -VNN which is located in the terminal 3 'part of each of the anchor primers, wherein V is a deoxyribonucleotide which is selected from the group consisting of A, C and G; and N is a deoxyribonucleotide which is selected from the group consisting of A, C, and T, the mixture includes anchor primers that contain all possibilities of V and N; (c) separating the population of double-stranded cDNAs with the first restriction endonuclease and a second restriction endonuclease, the second restriction endonuclease recognizes a sequence of four nucleotides to form a population of double-stranded cDNA molecules having a first and second terminal parts, respectively; (d) inserting each double-stranded cDNA molecule of step (c) into a vector in an orientation that is antisense to a T3 promoter within the vector to form a population of constructs containing the cDNA molecules inserted, by what are defined are 5 'and 3' flanking vector sequences adjacent to the 5 'terminal part of the direct strand of the inserted cDNA and the 3' terminal part of the direct strand, respectively, and such constructs have a lanting vector sequence 3 'of at least 15 nucleotides in length between the first restriction endonuclease site and a site defining the initiation of transcription in the promoter; (e) transforming Escherichia coli with the vector into which the separated DNA has been inserted to produce vectors containing cloned inserts; (f) generate linearized fragments containing the cDNA molecules inserted by digestion of the 'constructs produced in step (c) with at least one restriction endonuclease that does not recognize sequences in either the cDNA molecules inserted or in the T3 promoter; (g) generating a cRNA preparation of antisense cRNA transcript by incubating the linearized fragments with T3 RNA polymerase capable of initiating transcription from the T3 promoter; - (h) generating first strand cDNA by transcribing the cRNA using a reverse transcriptase and a 5 'RT primer having 15 to 30 nucleotides in length and comprising a nucleotide sequence that is complementary to the 5' flanking vector sequence; (i) generating a first set of PCR products by dividing the cDNA of the first chain in a first series of subacumulates and using the cDNA of the first chain as templates for a first polymerase chain reaction with a first PCR primer 3 'of 15 to 30 nucleotides in length which is complementary to the 3' flanking vector sequences, between the first restriction endonuclease site and a the site defining the initiation of transcription by the T3-specific promoter, and a first primer of 5 'PCR defined with a 3' terminal part consisting of Nx, where "N" "is one of the four deoxyribonucleotides A, C, G or T, the first 5 'PCR primer has 15 to 30 nucleotides of length and is complementary to the 5 'flanking vector sequence with the first complementary 5' PCR primer extending within a nucleotide of the specific nucleotides of cRNA insert, where a different one of the first 5 'PCR primers is used in each of the four different subacumulates; (j) generating a second set of PCR products by further dividing the first set of PCR products in each of a first series of subacumulates in a second set of subacumulates and using the first set of PCR products as templates for a second set polymerase chain reaction with a second 3 'PCR primer of 15 to 30 nucleotides in length that is complementary to the 3' flanking vector between the first restriction endonuclease site and the site defining the initiation of transcription by the T3 specific promoter , and a second 5 'PCR primer defined by a 3' terminal part consisting of Nx Nx, where Na is identical to the Nx used in the first polymerase chain reaction for this subacumulate, "N" is as in step (i), and "x" is an integer that is selected from the group consisting of 3 and 4, the primer is 15 to 30 nucleotides in length and is complementary to the 5 'flanking vector sequence. with primer complementarity extending through within the specific nucleotides of the cRNA insert in a number of nucleotides equal to "x" = 1, wherein a different one of the second 5 'PCR primers is used in different subacumulates of the second series of subacumulados and where there are 4X subacumulados in the second series of subacumulados for each of the subacumulados; (k) separating the second set of PCR products to generate a display of the sequence specific products representing the 3 'ends of the mRNAs present in the mRNA population. 49. The method of con? Dence with claim 48, characterized in that the mixture of 48 anchor primers have the sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 5). 50. The method according to claim 48, characterized in that the mixture of 48 anchor primers have the sequence G-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 8). 51. The method according to claim 48, characterized in that the mixture of 12 anchor primers have the sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 4). 52. The method according to claim 48, characterized in that the mixture of 12 anchor primers have the sequence G-A-T-T-T-C-A-C-T-G-G-A-G-C-G-G-C-C-C-G-C-A-G-G-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 7). 53. The method according to claim 48, characterized in that the mixture of 3 anchor primers have the sequence A-A-C-T-G-G-A-A-G-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V ('SEQ ID NO: 3). t 54. The method according to claim 48, characterized in that the mixture of 3 anchor primers has the sequence G-A-T-T-T-C-A-C-T-G-G-A-A-G-C-G-C-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 6). 55. The method according to claim 48, characterized in that the first restriction endonuclease is MspI and the second restriction endonuclease is No ti. 56. The method according to claim 48, characterized in that the first 5 'PCR primer is G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 22). 57. The con fi dence method with claim 48, characterized in that the first primer of PCR 3 'and the second PCR primer 3' are G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ID NO: 47). 58. The conformance method with claim 48, characterized in that the "x" in step (j) is 3. 59. The method according to claim 48, characterized in that the "x" in step (j) is 4. 60. A method for detecting a change in the pattern of mRNA expression in a tissue associated with a physiological or pathological change, characterized in that it comprises the steps of: (a) obtaining a first sample of a tissue that is not subject to physiological or pathological change; (b) isolating a population of mRNA from the first sample; (c) determining the mRNA expression pattern in the first tissue sample by performing steps (a) - (j) according to claim 1 to generate a first display of the specific sequence products representing the 3 'ends of the mRNAs present in the first sample; (d) obtaining a second sample of the tissue that has undergone the physiological or pathological change; (e) isolating a population of mRNA from the second sample; (f) determining the pattern of expression of mRNA in the second tissue sample when performing steps (a) - (j) according to claim 1, to generate a second display of the sequence specific products representing the ends 3 'of the mRNAs present in the second sample; and, (g) comparing the first and second displays to determine the effect of the physiological or pathological change in the pattern of mRNA expression in the tissue. 61. The method according to claim 60, characterized in that the physiological or pathological change is selected from the process mediated by transcription factors, intracellular second messengers, hormones, neurotransmitters, growth factors, neuromodulators, cell-to-cell contact, cell-to-cell contact. cell, cell to substrate contact, cell to extracellular matrix contact and contact between cell membranes and cytoskeleton. 62. The con fi dence method with claim 60, characterized in that the tissue is derived from the central nervous system. 63. The method of con fi dence with claim 62, characterized in that the physiological or pathological change is selected from the group consisting of Alzheimer's disease, parkinsonism, ischemia, alcohol addiction, drug addiction, schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, depression and bipolar manic depressive disorder. 64. The method according to claim 62, characterized in that the physiological or pathological change is associated with learning or memory, emotion, neurotoxicity to glutamate, feeding behavior, smell, vision, movement disorders, viral infection, electroshock therapy or the administration of a medicine or toxin. 65. The method according to claim 60, characterized in that the physiological or pathological change is selected from the group consisting of circadian variation, aging and long-term potentiation. 66. The method according to claim 60, characterized in that the tissue is derived from a structure within the central nervous system that is selected from the group consisting of retina, cerebral cortex, olfactory bulb, thalamus, hypothalamus, anterior pituitary, posterior pituitary, hippocampus , nucleus acombens, amygdala, etriatus, cerebellum, brain stem, suprachiasmatic nucleus and spinal cord. 67. The method according to claim 60, characterized in that the tissue is normal or neoplastic tissue of an organ or organ system that is selected from the group consisting of the cardiovascular system, the pulmonary system, the digestive system, the peripheral nervous system, the liver, kidney, skeletal muscle and the reproductive system. 68. The method according to claim 60, characterized in that the tissue is normal or neoplastic tissue comprising cells that are taken or that are derived from an organ or organ system that is selected from the group consisting of the cardiovascular system, the lymphatic system , the respiratory system, the digestive system, the peripheral nervous system, the central nervous system, the enteric nervous system, the endocrine system, the integument (which includes skin, hair and nails), the skeletal system (which includes bone and muscle) , the urinary system and the reproductive system. 69. The method according to claim 60, characterized in that the tissue is normal or neoplastic tissue comprising the cells that are taken or derived from the group consisting of epithelium, endothelium, mucosa, glands, blood, lymph, connective tissue, cartilage, bone , smooth muscle, skeletal muscle, cardiac muscle, neurons, glia cells, spleen, thymus, pituitary, thyroid, parathyroid, adrenal cortex, adrenal medulla, adrenal cortex, pineal, skin, hair, nails, teeth, liver, pancreas, lung , kidney, bladder, ureter, breast, ovary, uterus, vagina, testicle, prostate, penis, eye and ear. 70. "A method for detecting a difference in the action of a drug to be examined and a known compound, characterized in that it comprises the steps of: (a) obtaining a first tissue sample from an organism treated with a compound of known physiological function (b) ^ isolate a population of mRNA from the first sample; (c) determine the pattern of mRNA expression in the first tissue sample when performing steps (a) - (j) of claim 1 to generate a first display of sequence-specific products representing the 3 'ends of the mRNAs present in the first sample, (d) obtaining a second tissue sample from an organism treated with a drug to be examined for a difference in the action of the drug and the known compound, (e) isolating a population of mRNA from the first sample, (f) determining the pattern of mRNA expression in the second tissue sample by performing steps (a) - (j) of in accordance with claim 1, to generate a second display of the sequence specific products representing the 3 'ends of the mRNAs present in the second sample; and (g) comparing the first and second displays in order to detect the presence of mRNA species whose expression is not affected by the known compound but which is affected by the drug to be examined, thus indicating a difference in the action of the drug to be examined and the known compound. 71. The method according to claim 70, characterized in that the medicament to be examined is selected from the group consisting of antigens, antigens, anticonvulsants, monoamine inhibitors. oxidase and stimulants. 72. The method according to claim 70, characterized in that the medicament to be examined is selected from the group consisting of antiparkinsonian agents, skeletal muscle relaxant, analgesics, local anesthetics, cholinergics, antiviral agents, antispasmodics, steroids and drugs. Nonsteroidal anti-inflammatory drugs. 73. A database, characterized in that it comprises the data produced by the quantification of the display of the specific sequence products according to claim 1. 74. The database according to claim 1, characterized in that it also comprises data regarding sequence relationships, gene mapping and cell distributions. 75. A method for recognizing sequence identities and similarities between the sequence of the 3 'ends of mRNA molecules present in a sample and a sequence database, characterized in that it comprises the steps of: (a) preparing a population of chain cDNA doubling from a population of mRNA using a mixture of anchor primers, each anchor primer has a 5 'terminal part and a 3' terminal part and includes: (i) a stretch of 7 to 40 T residues; (ii) a site for separation by a first restriction endonuclease that recognizes more than six bases, the site for separation is located toward the 5 'end portion relative to the T residue portion; (iii) a first filler segment of 4 to 40 nucleotides, the first filler segment is located towards the 5 'end portion relative to the site for separation by the first restriction endonuclease; (iv) a second filler segment interposed between the site for separation by a first restriction endonuclease that recognizes more than six bases and the stretch for residues T, and (v) phase adjustment residues that are located at the terminal 3 'part of each of the anchor primers "which are selected from the group consisting of -V, -VN, and -VN-NJ wherein V is a deoxyribonucleotide which is selected from the group consisting of A, C and G; is a deoxyribonucleotide that is selected from the group consisting of A, C, G and T, the mixture includes anchor primers that contain all possibilities for V and N; '(b) separating the population of double-stranded cDNA with the first restriction endonuclease and a second restriction endonuclease, the second restriction endonuclease recognizes a sequence of four nucleotides to form a population of double-stranded cDNA molecules having first and second terminal portions, respectively; ) inserting each double-stranded cDNA molecule from step (b) into a vector in an orientation that is antisense to the bacteriophage-specific promoter within the vector to form a population of constructs containing the inserted cDNA molecules, thus that 5 'and 3' flanking vector sequences are defined adjacent to the 5 'end portion of the direct (sense) strand of the inserted cDNA, and the 3' end portion of the direct strand, respectively, and the constructs have a sequence of 3 'flanking vector of at least 15 nucleotides in length between the first restriction endonuclease site and a site defining the start of transcription in the promoter; (d) transforming a host cell with the vector into which the separated cDNA has been inserted, to produce vectors containing cloned inserts; (e) generating linearized fragments containing the cDNA molecules inserted by digestion of the constructs produced in step (d) with at least one restriction endonuclease that does not recognize sequences in either the cDNA molecules inserted or in the specific promoter of bacteriophage, but which recognizes sequences in the vector, so that the resulting linearized fragments have a 5 'flanking vector sequence of at least 15 nucleotides within the 5' vector relative to the second terminal part of the cDNA molecules of double chain; (f) generating a mRNA preparation of nonsense cRNA transcripts by incubating the linearized fragments with a bacteriophage-specific RNA polymerase capable of initiating transcription from the bacteriophage-specific promoter; (g) generating first-strand cDNA by transcribing the cRNA using a reverse transcriptase and a 5 'RT primer having 15 to 30 nucleotides in length and comprising a nucleotide sequence that is complementary to the 5' flanking vector sequence; (h) generating a first set of PCR products by dividing the cDNA of the first strand in a first set of subacumulates and using the cDNA of the first strand as templates for a first polymerase chain reaction with a first PCR primer. 'from 15 to 30 nucleotides in length which is complementary to the 3' flanking vector sequences between the first restriction endonuclease site and the site defining the initiation of transcription by the bacteriophage-specific promoter and a first 5 'PCR primer. defined with a 3 'terminal part consisting of N17 where "N" is one of the four deoxyribonucleotides A, C, G or T, the first 5 'PCR primer has 15 to 30 nucleotides in length and is complementary to the 5 'flanking vector sequence with the first of the 5' complementary PCR primers extending within a nucleotide of the insert specific nucleotides of the cRNA, where "a different one from the First 5 'PCR primers are used in each of the four different subacumulates: (i) generate a second set of PCR products by further dividing the first set of PCR products in each of the first series of subacumulates in a second series of sub-cumulatives and use the first set of PCR products as templates for a second polymerase chain reaction, with a second 3 'PCR primer of 15 to 30 nucleotides in length that is complementary to the 3' flanking vector sequences between the first restriction endonuclease site and the site that defines the start of transcription by a bacteriophage-specific promoter, and a second 5 'PCR bator defined with a 3' terminal part consisting of -N1 -'NX, where Nx is identical to N? which is used in the first polymerase chain reaction for that subacumulate, "N" is as in step (h) and "x" is an integer from 1 to 5, the primer is 15 to 30 nucleotides in length. and complementary to the 5 'flanking vector sequence with primer complementarity extending through the specific nucleotides of 'Yes insert, the cRNA in a number of nucleotides equal to "x" + 1, where, a different one of the second 5' PCR primers are used in different subacumulates of the second series of subacumulates where there are 4X subacumulated in the second series of subacumulados for each of the subacumulados in the first set of subacumulados; (j) separating the second set of PCR products to generate a display of sequence specific products representing the 3 'ends of the mRNAs present in the mRNA population; (k) eluting at least one cDNA corresponding to a MRNA from an electropherogram in which bands representing the 3 'ends of the mRNA present in the sample are displayed / (1) amplify the cDNA eluted in a polymerase chain reaction; (m) cloning the amplified cDNA in a plasmid; (n) producing DNA corresponding to the cloned DNA of the plasmid; determine the sequence of the cloned cDNA; determining the corresponding nucleic sequences from a database of the * nucleotide sequences, the corresponding nucleotide sequences are bounded by the most distal recognition site for the second endonuclease and the start of the poly (A) tail; and (q) comparing the sequence of the cloned cDNA with the corresponding nucleotide sequences whereby identities and sequence similarities are recognized between the sequence of the 3 'ends of the mRNA molecules present in a sample and a database of the sequences . 76. The method according to claim 75, characterized in that it further comprises the step of: (r) comparing the length and quantity of the PCR products in a two-dimensional graphical display. 77. The method according to claim 76, characterized in that it further comprises the steps of: (s) determining the expected length of the corresponding nucleotide sequence, which is equal to the sum of the lengths of the corresponding nucleotide sequence determined from the database, the length of the 5 'PCR sequence hybridizable to the vector sequence, the length of the anchor primer sequence remnant, and the interposed segment of the vector sequence and the length of the 3 'PCR sequence that can be hybridized to the sequence of the vector; and (t) comparing the length of the PCR product with the determined expected length of the corresponding nucleotide sequence, wherein the expected length of the corresponding nucleotide sequence is indicated by the two-dimensional graphical display by use of a graphic symbol or text character . 78. A method for recognizing sequence identities and similarities between the sequence of a cDNA fragment corresponding to a mRNA molecule present in a sample and a sequence database, characterized in that it comprises the steps of: eluting a corresponding cDNA fragment to a mRNA molecule present in a sample; amplifying the cDNA fragment eluted in a polymerase chain reaction to produce an amplified cDNA fragment; cloning the amplified cDNA fragment into a plasmid; producing a DNA molecule corresponding to the cloned cDNA fragment; sequencing the produced DNA molecule, whereby the sequence of the eluted cDNA fragment is determined; and comparing the sequence of the cDNA fragment eluted with the sequences in a database whereby identities and sequence similarities are recognized. 79. The method according to claim 78, characterized in that the step of comparing the sequence of the cDNA fragment eluted with the sequences in a database is performed using a computer. 80. The method according to claim 78, characterized in that it comprises the additional step of graphically displaying the results of the comparison. 81. A method for recognizing sequence identities and similarities between the sequence of a cDNA fragment corresponding to a mRNA molecule present in a sample and a sequence database, characterized in that it comprises the steps of: eluting a corresponding cDNA fragment with a mRNA molecule present in a sample, wherein the cDNA fragment has a length determined by the position of a restriction endonuclease recognition site and a poly (A) tail of the mRNA molecule; determining a partial sequence of the cDNA fragment by performing a polymerase chain reaction with a 5 'PCR primer corresponding to the sequence of the restriction endonuclease recognition site, and comparing the determined partial sequence of the eluted cDNA fragment and the length of the cDNA fragment cbn the sequences in a database so identities and sequence similarities are recognized. 82. A method for producing a transformed polynucleotide sequence database entry, characterized in that it comprises the steps of: choosing a source sequence from a polynucleotide sequence database entry; locate a poly (A) tail sequence within the source sequence; locating a sequence of the endonuclease recognition site within * the source sequence that is closest to the first recognition site; determining an index sequence consisting of about two to about six nucleotides adjacent to the endonuclease recognition site; determining a correlated sequence within a source sequence, the correlated sequence includes the sequence linked by the poly (A) tail and the endonuclease recognition site and includes at least part of the endonuclease recognition site; determine the length of the correlated sequence; and i store information regarding the location and sequence of the poly (A) tail, the location and sequence of the endonuclease recognition site, and the length of the sequence correlated in relation to the source sequence, whereby an input is produced of transformed database. 83. The method according to claim 82, characterized in that it further comprises the step of: graphically displaying the length of the sequence correlated in relation to the index sequence. 84. The method according to claim 83, characterized in that the restriction endonuclease is selected from the group consisting of MspI, Taql and HinPlI. 85. A method for improving the resolution of the length and quantity of PCR products by decreasing the background that is due to the amplification of the non-targeted cDNAs, characterized in that it comprises the steps of: Selecting a sample from a population of cRNA, wherein each cRNA molecule comprises inserting a sequence and a sequence derived from vector; performing reverse transcription using a reverse transcription primer that hybridizes to the vector-derived sequence and extends from about 5 nucleotides to about 6 nucleotides within the insert sequence to produce a reverse transcription product of cDNA; subdivide the reverse transcription product of cDNA; performing at least one polymerase chain reaction using the subdivided cDNA reverse transcription product, a 3 'PCR primer and a 5' PCR primer that hybridizes to the vector-derived sequence and extends from about 7 nucleotides to about 9 nucleotides within the insert sequence to produce a PCR product, whereby the background is decreased which is due to the amplification of the non-targeted cDNAs. 86. The method according to claim 85, characterized in that there are 16 accumulated reverse transcription reactions and there are 16 different reverse transcription primers. 87. The method according to claim 86, characterized in that there are 4X subacumulated polymerase chain reactions, where X is the difference between the number of nucleotides extending the 5 'PCR primer within the insert sequence and the number of nucleotides extending the reverse transcription primer within the insert sequence. 88. A method for sequence-specific simultaneous identification of mRNAs in a population of mRNA comprising the steps of: (a) preparing a population of double-stranded cDNA from a population of mRNA using a mixture of anchor primers, each primer The anchor has a terminal part 5 'and a terminal part 3' and includes: (i) a stretch of 7 to 40 residues T; (ii) a site for separation by a first restriction endonuclease that recognizes more than six bases, the site for separation is located towards the 5 'end portion relative to the stretch of the T residues; (iii) a first filler segment of 4 to 40 nucleotides, the first filler segment is located towards the 5 'end portion relative to the site for separation by the first restriction endonuclease; (iv) a second filler segment interposed between the site for separation by a first restriction endonuclease that recognizes more than six bases and the tract of T residues, and (v) phase adjustment residues that are located in the 3 'terminal part of each of the anchor primers that are selected from the group consisting of -V, -VN, and -VNN, wherein V is a deoxyribonucleotide that is selected from the group consisting of A, C and G; and N is a deoxyribonucleotide which is selected from the group consisting of A, C, G and T, the mixture includes anchor primers containing all possibilities for V and N; , (b) separating the population of double-stranded cDNA with the first restriction endonuclease and a second restriction endonuclease, the second restriction endonuclease recognizes a sequence of four nucleotides, to form a population of double-stranded cDNA molecules; (c) inserting each double-stranded cDNA molecule from step (b) into a vector containing a bacteriophage-specific promoter within the vector to form a population of constructs containing the double-stranded cDNA molecules "inserted, so that a first r flanking vector sequences of at least 15 nucleotides in length are defined from a site defining the start of transcription in the promoter to the first inserted cDNA terminal part and a second flanking vector sequence in at least 15 nucleotides in length from a restriction endonuclease site that is not present in either the inserted cDNA molecule or the bacteriophage-specific promoter and extends to the second terminal part of the cDNA molecules; (d) transform a cell host with the vector into which the separated cDNA has been inserted to produce vectors containing cloned inserts; (e) generate linearized fragments containing the double-stranded cDNA molecules inserted by digestion of the constructs produced in step (d) with at least one restriction endonuclease that does not recognize sequences either inserted into the cDNA molecules or into the specific promoter of bacteriophage, but sequences are recognized in the vector, such that the resulting linearized fragments have a bacteriophage-specific promoter with a first 5 'flanking vector sequence of at least 15 nucleotides within the 5' vector for the second terminal part of the cDNA molecules inserted, and a second flanking vector sequence of at least 15 nucleotides in length extending beyond the second terminal part of the cDNA molecule; (f) generating a cRNA preparation of cRNA transcripts by incubating the linearized fragments with a bacteriophage-specific RNA polymerase capable of initiating transcription from the bacteriophage-specific promoter; (g) generating first-strand cDNA by transcribing the cRNA using a reverse transcriptase and an RT 5 'primer that is 15 to 30 nucleotides in length and that is complementary to the sequence of cRNAs derived from the second-sequence of the flanking vector; (h) generating a first set of PCR products by dividing the cDNA of the first strand in a series of subacumulates and using the cDNA of the first strand as templates for a first polymerase chain reaction with a first 3 'PCR primer. 15 to 30 nucleotides in length which is complementary to the first 3 'flanking vector sequences between the first restriction endonuclease site and the site defining the initiation of transcription by the bacteriophage-specific promoter and a first 5' PCR primer defined with a 3 'terminal part consisting of -Nlr where "N" is one of the four deoxyribonucleotides A, C, G, or T, the first 5' PCR primer is 15 to 30 nucleotides in length and is complementary to the 5 'flanking vector sequence with the first complementary 5' PCR primers extending into a nucleotide of the specific nucleotides of the cRNA insert, wherein a different one of the first PC primers R 5 'are used in each of the four different subacumulates; (i) generate a second set of PCR products by further dividing the first set of PCR products in each of the first series of sub-cumulatives in a second set of subacumulates and using the first set of PCR products as templates for a second set polymerase chain reaction with a second 3 'PCR primer of 15 to 30 nucleotides in length that is complementary to the first 3' flanking vector sequences between the first restriction endonuclease site and the site defining the initiation of transcription by the bacteriophage specific promoter, and a second 5 'PCR primer defined with a 3' terminal part consisting of Nx Nx, where NL is identical to the Nx used in the first polymerase chain reaction for that subacumulate, "N" is as in step (h) and "x" is an integer from 1 to 5, the primer is 15 to 30 nucleotides in length and complementary to the second flanking vector sequence with the primer complementarity extending through the specific nucleotides of the cRNA insert in a number of nucleotides equal to "x" + 1, wherein a different one of the second 5 'PCR primers are used in different subacumulates of the second series of subacumulates where there are 4X subacumulados in the second series of subacumulados for each of the subacumulados in the first set of subacumulados; and (j) separating the second set of PCR products to generate a display of sequence specific products representing the 3 'ends of the mRNAs present in the mRNA population.