WO1999042610A1 - VERFAHREN ZUR CHARAKTERISIERUNG VON mRNA-MOLEKÜLEN - Google Patents
VERFAHREN ZUR CHARAKTERISIERUNG VON mRNA-MOLEKÜLEN Download PDFInfo
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- WO1999042610A1 WO1999042610A1 PCT/EP1999/000992 EP9900992W WO9942610A1 WO 1999042610 A1 WO1999042610 A1 WO 1999042610A1 EP 9900992 W EP9900992 W EP 9900992W WO 9942610 A1 WO9942610 A1 WO 9942610A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
- C12Q1/6855—Ligating adaptors
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
Definitions
- the present invention relates to methods for the qualitative and quantitative detection of differentially expressed mRNA molecules.
- the technology on which the invention is based is briefly referred to below as DEPD (Digital Expression Pattern Display).
- the genome of higher organisms comprises around 100,000 different genes, of which only a comparatively small number are expressed in every cell of an organism and thus converted into polypeptides and proteins. It is assumed that to a large extent all processes and metabolic performance in the area of living matter depend on which genes are switched on and off at what time in soft tissues. Numerous findings indicate that cellular processes such as homeostasis, reactions to allergies, regulation of the cell cycle, aging and the entry of cells into programmed cell death (apoptosis) are based on, or are related to, the differential expression of certain genes. Both the course of normal development and that of diseases such as Cancer-causing pathological phenomena are essentially due to changes in gene expression.
- the method according to the invention is used, in particular, to record as much as possible of all mRNA molecules present in a cell or in a tissue and to compare them with other rows of tissues or with specific conditions (disease or development stages) or treatment phases thereof, preferably both in qualitative and in terms of treatment in quantitative terms.
- the method according to the invention thus allows, for example, the creation of a comprehensive image of the various mRNA molecules present in a defined mRNA population and the subsequent use of the preferably digital information obtained in the context of - 2 -
- the present method therefore allows the holistic detection and characterization of cellular processes that are reflected in specific expression patterns at the level of the mRNA populations. In this way, for example, changes in the expression pattern of individual genes involved in a specific process can be identified quickly and reliably. In this way, new active substances for active pharmaceutical ingredients can be defined.
- the information obtained can also be used to establish the causal relationship between known target genes and target proteins via traceable biochemical signaling or synthetic routes.
- P. Liang and AB Pardee describe a method for the separation of individual mRNAs by means of polymerase chain reaction (PCR) (P. Liang & AB Pardee 1992 Science 257, 967-971). This method was used to compare the mRNA populations expressed by two related cell types.
- PCR polymerase chain reaction
- the products were separated on sequencing gels and 50-100 bands in the size range of 100-500 nucleotides were observed.
- the bands resulted from the amplification of cDNAs corresponding to the 3 ' ends of mRNAs containing the complement of the 3' anchor primer and the arbitrarily chosen 5 'oligomer.
- the patterns of the bands amplified from both cDNAs were similar for each pair of primers, the intensities of approximately 80% of the bands being indistinguishable. Certain bands appeared in one or the other PCR - 3 -
- WO 95/13369 discloses a method (TOGA - TOtal Gene Expression Analysis) for the simultaneous identification of differentially expressed mRNAs and for measuring their relative concentrations.
- the method is based on the generation of double-stranded cDNA from isolated mRNA using a specific set of oligo (dT) primers.
- a mixture of 12 anchor primers with the following structure is used: starting from 5 ', a "stuffer” or “heel” fragment of 4-40 bases is followed by a recognition sequence for a restriction endonuclease (typically NotI), 7-40 dT nucleotides and finally two “anchor bases” V, N at the 3 'end of the primer.
- V denotes a deoxyribonucleotide from group dA, dC or dG
- N defines the deoxyribonucleotides dA, dC, dG and dT.
- the cDNA obtained in this way is then completely digested with a restriction enzyme which recognizes 4 bases as the sequence for the cleavage (eg Mspl), cut with NotI and cloned into a correspondingly treated plasmid vector.
- the orientation of the insert is "antisense" relative to a vector-encoded, bacteriophage-specific promoter (typically T3).
- the ions are transformed into an E.coii strain, which generates cDNA banks.
- the plasmid DNA of these cDNA libraries is isolated and linearized using combination digests by 6 different restriction enzymes, which are different from those used above.
- the linearized cDNA is translated into cRNA by the T3 polymerase and then transcribed into 16 sub-fractions of single-stranded cDNA.
- a thermostable reverse transcriptase at high temperature and one of 16 different cRNA primers, the two 3'-nucleotides of which consist of a complete permutation of the 4 possible deoxyribonucleotides dA, dC, dG and dT, are used.
- the products of the 16 cDNA fractions are used as templates for PCR using a 3'-oligonucleotide, which is one Vector sequence corresponds to the cloning site of the insert, and a 5 ' oligomer which corresponds to one of the 16 cDNA synthesis primers with an additional two 3 ' nucleotides of the complete permutation of the 4 possible deoxyribonucleotides dA, dC, dG and dT.
- up to 256 different pools are generated, whose radiolabelled bands (35S-dATP or 32P-dCTP) are analyzed on polyacrylamide gels.
- radiolabelled bands 35S-dATP or 32P-dCTP
- thermostable reverse transcriptase for the permuted primers which are used for the second cDNA synthesis, since the selectivity of the reverse transcriptase in the case of base mismatch is generally approx. 10-1,000 times (for AMV-RT) below the selectivity the Taq polymerase (LN. Mendelman et al. 1990, J. Biol. Chem. 265, 2338-2346);
- double-stranded cDNA is generated using an oligonucleotide of the structure "Heel-dT (13)", "Heel” being a sequence of 12 bases.
- the cDNA is completely digested by a restriction enzyme with a 4-base recognition sequence (Rsal) and ligated with a "pseudo double-strand adapter".
- This molecule consists of a longer (39 bases) and a shorter (12 bases complementary to the 3 ' end of the longer) oligomer, which are hybridized against one another under suitable conditions.
- the 5 'ends of the oligonucleotides are not phosphorylated.
- the cDNA synthesis primer with an additional two 3'-nucleotides of the complete permutation of all four deoxyribonucleotides dA, dC, dG and dT and a primer which is the 3 ' end of the longer adapter oligomer with an additional two 3' nucleotides the complete permutation of all four deoxyribonucleotides dA, dC, dG and dT is used.
- an artificial mismatch was introduced into the oligonucleotide at position -4 relative to the 3 'end of the primer.
- Y. Prashar and S. Weissman (1996, Proc. Natl. Acad. Sci. USA 93, 659-663) describe a process in which double-stranded cDNA is produced by means of 12 oligonucleotides which have the following structure: one starting from 5 'follows one "Heel” structure is a sequence of 18 dT nucleotides and two "anchor bases" V, N at the 3 ' end of the primer.
- V denotes a deoxyribonucleotide of the group dA, dC or dG
- N defines the deoxyribonucleotides dA, dC, dG and dT.
- the cDNA synthesis takes place at a temperature of 50 ° C and should enable the complex mixture to be divided into 12 different pools.
- the resulting DNA fragments are provided with an adapter, which has the structure of a letter.
- an oligonucleotide is used as the 5 ' primer, the binding site of which lies in the outer region of the Ypsilon adapter and which only arises when the complementary strand to this region is formed in a first synthesis. All fragments that have a Ypsiion adapter on both sides cannot be amplified.
- WO 97/2921 1 describes the 'restriction display (RD-PCR)' technique, in which double-stranded cDNA is produced using 12 oligonucleotides.
- These primers have the following structure: starting from 5 ', a "heel” structure is followed by two deoxynucleotides of the complete permutation of all four deoxyribonucleotides dA, dC, dG and dT, a sequence of 17 dT nucleotides and two "anchor bases" V, N am 3 'end of the primer.
- V denotes a deoxyribonucleotide from group dA, dC or dG, while N denotes - 7 -
- Deoxyribonucleotides dA, dC, dG and dT defined. After complete digestion of the cDNA with one or more restriction endonucleases, an adapter molecule is ligated to the cDNA fragments. In a subsequent PCR, a 3'-primer is used which binds selectively to the "heel" structure of the cDNA and additionally has two 3'-nucleotides V, N at the 3 ' end of the primer.
- V denotes a deoxyribonucleotide from group dA, dC or dG, while N defines the deoxyribonucleotides dA, dC, dG and dT.
- oligonucleotide corresponding to the 3 'sequence of the adapter primer is used as the 5' primer, which additionally contains a 3 'nucleotide or two 3' nucleotides or three 3 'nucleotides for the complete permutation of all four deoxyribonucleotides dA, dC, dG and dT.
- the PCR is carried out with different permutation combinations in such a way that in a first PCR (or in the first 10-25 PCR cycles) 5 'primers with only one permutation and then in a second PCR (or in the remaining PCR Cycles) 5 'primers with only two or three permutations can be used. This is said to significantly increase the selectivity for the various 5'-primer permuations.
- Kato describes a method ('molecular indexing' - 1995, Nucl. Acids Res., Vol. 23, 3685-90 and 1996, Nucl. Acids Res., Vol. 24, 394-95), which is based on the digestion of the double-stranded cDNA with restriction endonucleases of class IIS. These generate 5 'overhangs of the cDNA of unknown sequence.
- 64 biotinylated adapters, the nucleotides of which 2-4 (relative to the 5 'end) of their 5' overhangs are complementary to a 64 step of the entire cDNA pool, are then ligated with DNA ligase from E. coli.
- the respective 5 'nucleotide of the adapter overhangs remains undefined.
- the cDNA fragments ligated in this way are purified by binding to streptavidin-coupled magnetic particles.
- the adapter-ligated 3 'ends of the cDNA are removed using an adapter oligonucleotide and an oligo (dT) oligomer which is attached to the 3' - 8th -
- a method for identifying and characterizing mRNA molecules comprises the following steps:
- step (b) wherein for the 1st and 2nd alternatives in step (b) the synthesis of the first strand cDNA molecules takes place by reverse transcription using an anchored oligo-dT nucleotide which has a 3'-extension of 2 bases, the first base dA , dC or dG, and the second base is dA, dC, dG or dT, and which has a 5 'extension of 5-15, preferably 6-15, bases which are suitable for the - 10 -
- step (b) Encoding a restriction endonuclease site with the cleavage characteristic 16/14 'downstream' of the recognition sequence; or wherein for the third alternative in step (b) the cDNA first strand molecules are synthesized by reverse transcription using an anchored oligo-dT nucleotide which has a 3'-extension of 2 bases, the first base dA, dC or dG, and the second base is dA, dC, dG or dT, and which has a 5 'extension of 5-15 bases of any sequence.
- Enzymes with the cleavage characteristic 16/14 "downstream" of the recognition sequence are known to the person skilled in the art. Examples are Eco571 and Bsg1 (see, for example, A. Janulaitis et al., Nucleic Acids Res. 20 (1992), pp. 6043-6049; Petrusyte et al., Gene 74 (1988), pp. 89-
- the oligo-dT nucleotide is preferably completely substituted by 2'-O-methylated ribonucleotides.
- the oligo-dT nucleotide can consist of standard deoxyribonucleotides.
- the oligo-dT nucleotide is provided with a biotin residue at its free 5 'end and / or at internal dT nucleotides via a C9 spacer.
- a preferred embodiment relates to a method according to the invention, wherein in step (h) of the second alternative or in step (e) of the third alternative, the mismatches at positions -3, or -3 and -4, or -4 and -5 compared to the complementary strand determined by the adapter molecules.
- a further preferred embodiment of the method according to the invention is characterized in that a restriction enzyme of class IIS is used in step (c) which has 5 nucleotides as the recognition sequence and one of 2-4, in particular 4, nucleotides which are not part of the recognition sequence, existing overhang of the cut cDNA fragments generated.
- a restriction enzyme of class IIS is used in step (c) which has 5 nucleotides as the recognition sequence and one of 2-4, in particular 4, nucleotides which are not part of the recognition sequence, existing overhang of the cut cDNA fragments generated.
- a further preferred embodiment of the method according to the invention is characterized in that in step (f) of the 1st alternative or in step (e) of the 2nd alternative the - 11 -
- step (g) of the 1st alternative or in step (f) of the 2nd alternative of the method according to the invention a restriction enzyme of class IIS is used.
- step (d) or from step (h) of the first alternative or from step (g) of the second alternative of the method according to the invention prior to the amplification according to step (i) with a nuclease, selected from the group, are further preferred from T4 endonuclease VII, S1 nuclease, and mung bean nuclease.
- the method according to the invention is preferably characterized in that in step (i) of the first alternative or in step (h) of the second alternative or in step (e) of the third alternative, the cDNA fragments are amplified using oligonucleotides, that hybridize to the complementary strand of the sense oligomer of the ligated adapter molecules.
- the method according to the invention is further preferably characterized in that in step (k) of the first alternative or in step (j) of the second alternative or in step (g) of the third alternative, the analysis is based on the different lengths of the products and in knowledge of the by manipulation known base sequence of 9 or 10 nucleotides.
- a further preferred embodiment relates to the use of the method according to the invention according to one of the preceding claims for possibly computer-aided identification and isolation and analysis of new genes.
- the method DEPD according to the invention was developed in order to significantly reduce the error rate in the identification of some nucleotides of the cDNA fragments, which in turn permit the identification of the encoded gene in a suitable database.
- the improved performance of the method according to the invention results from the use of specific ligation techniques in a suitable combination with permutation-specific mismatch PCR. After selective purification of the 3 'ends of the cDNAs, an adapter molecule is ligated to the 5' and 3 'ends of the fragments. In the following - 12 -
- PCR primers are used which preferably each have two bases as permutation.
- the permutation specificity of the PCR primers can be significantly increased at a high annealing temperature and preferably with the simultaneous introduction of artificial template mismatches at selective oligonucleotide sites.
- an error-correcting effect is obtained by using permutation primers on both sides of the cDNA template, since such amplification products, which were erroneously created in one of the first rounds of PCR by means of an oligomer, in their further propagation can be suppressed if the counter-primer only amplifies the correct permutation.
- the suitable, combined use of ligation and PCR permutation technology in the DEPD method makes it possible to define 9 or 10 nucleotides and the length of an amplified fragment. This information should also be sufficient for the reliable identification of a gene, preferably by means of database analysis, if there should be an error in determining the base sequence of the cDNA fragment.
- the use of the DEPD method according to the invention enables, among other things, a comprehensive analysis of the interaction of all genes involved in a defined system and / or in a defined situation at the level of the mRNA expression pattern, with only small amounts of tissue or cells being required for specific and reproducible results become.
- the method can be used in a variety of applications. This includes, for example, the comparison of organs, tissues, tissue parts, or diseased tissues or tissue parts with appropriate healthy material, possibly also in the context of a comparative investigation using active pharmaceutical ingredients against corresponding controls - 13 -
- the method according to the invention enables a comparison of defined states in animal models, with comparative analyzes of organs, tissues, tissue parts or diseased tissues or tissue parts being preferred over corresponding healthy material. Further areas of application concern the analysis of transgenic animals, which also include so-called ' knock-out ' animals, as well as the phenotypic evaluation of the use of antibodies, antisense and ribozyme oligonucleotides and comparable agents, which are used in the context of functional approaches to elucidate the relevance certain genes are used.
- Another aspect of the present invention is its preferred use in the context of a database-oriented gene expression analysis method.
- the method according to the invention represents a surprisingly simple and inexpensive alternative to the known methods of the type described above in terms of quality is clearly superior.
- RNA isolation and purification of total RNA from tissues to be examined, tissue parts, biopsy samples, cells etc. is carried out according to the standard methods described.
- an enzymatic digestion is carried out with DNasel (Boehringer Mannheim, Germany).
- the polyA + mRNA is then purified from the total RNA by means of oligo (dT) -coupled magnetic particles (Oligo (dT) magnetic beads, Promega, Wl, USA).
- Double-stranded cDNA is synthesized from mRNA using a mixture of 12 anchor primers with the following structure: starting from 5 'is followed by a "HeeP" fragment (5 "extension) of 5-15 bases, for example 5 or 6 bases Detection sequence for one of the restriction enzymes Bsgl or Eco57l. This is followed by a sequence of 14 dT nucleotides and the two anchor nucleotides V, N at the 3 'end of the primer.
- V denotes a deoxyribonucleotide of the group dA, dC or dG
- N defines the deoxyribonucleotides dA, dC, dG and dT
- the deoxyribonucleotides of the dT sequence possibly also of the "Heel" fragment, are completely substituted by 2'-O-methylated ribonucleotides 5'-end and / or on one or more internal dT nucleotides with a biotin residue (C9 spacer).
- cDNA synthesis can be carried out with a mixture of 12 unmodified, i.e. anchor primers consisting of deoxyribonucleotides are carried out.
- First adapter ligation a) The enzyme digestion creates a 5 'overhang of four unknown nucleotides in the cDNA. Sixteen adapter molecules consisting of two oligomers (not 5 ' - phosphorylated, "pseudo double-stranded" adapters) are ligated to the cDNA after restriction digestion has taken place.
- the adapter typically consists of two oligonucleotides of different lengths, the longer of 25-35, the shorter (complementary to the 3 ' end of the longer) of 8-25 bases, in particular 8-12 bases.
- the adapter is created by hybridizing the two oligonucleotides against each other under suitable conditions and forms a 4-nucleotide overhang, with nucleotide 3 or 4 ('inner' nucleotides - relative to the 5 'end of the antisense oligomer) one of the four possible deoxyribonucleotides dA, dC, dG and dT can be, while the 'outer' nucleotides 1 and 2 (relative to the 5 'end of the antisense oligomer) remain undefined, ie they always consist of a mixture of all four deoxyribonucleotides.
- the ligation batches are subsequently incubated with a nuclease selected from the group consisting of T4 endonuclease VII, S1 nuciease and mung bean nuclease.
- the 16 permuted adapter molecules are ligated at 4-37 ° C., 1-16 hours, 0-150 mM Na acetate, 1-5 units of T4 DNA ligase (or Taq DNA ligase or E. coli DNA ligase) in a suitable buffer. After purification, each of the 16 ligation batches is used as a template in a PCR (see (8a)).
- coli DNA ligase is able to discriminate the first three bases of the adapter overhang in the ligation. This takes place under the conditions already described above. To ensure the correct adapter ligation, the ligation approaches are subsequently selected with a nuclease from the - 16 -
- T4 endonuclease VII Group consisting of T4 endonuclease VII, S1 nuclease, and mung bean nuclease.
- the 5 overhangs of the cDNA are successively filled with deoxyribonucleotides using Kienow DNA polymerase. This is possible through the use of dideoxyribonucleotides (ddNTP) and competitive adapter molecules.
- ddNTP dideoxyribonucleotides
- competitive adapter molecules the 3 'ends of the cDNA are not eluted from the magnetic particles in order to be able to remove the nucleotides used after each step.
- the cDNA is first incubated with ddC nucleotides in order to block all overhangs which begin with a dG. After removal of the didesxoribonucleotides, the cDNA is incubated with dA nucleotides. This is followed by the ligation of an adapter moiecule, which has a 'biunt' and a 'sticky' end, to those cNDAs which have until then a filled 5 'overhang. Two competitive adapter molecules are ligated in the same step.
- the 3 'ends of the cDNA can preferably be selectively purified from the cDNA mixture with the aid of magnetic particles (magnetic beads) which are coupled to streptavidin (see also Biomagnetic Techniques in Molecular Biology, Dynal, N-0212 Oslo, Norway). This type of purification ensures that in the subsequent PCR there is no unspecific amplification of internal (ie non-3 ' ends) cDNA fragments.
- the cDNA is eluted from the magnetic particles either by extraction with organic solvents at high temperature - 17 -
- the adapter typically consists of two oligonucleotides of different lengths, the longer of 25-35, the shorter (complementary to the 3 ' end of the longer, 5'-phosphorylated) consisting of 22-30 bases.
- the adapter is created by hybridizing the two oligonucleotides against each other under suitable conditions and forms a 2-nucleotide overhang, the 'outer' 3 'nucleotide being one of the four deoxyribonucleotides dA, dC, dG or dT, while the' inner 'nucleotide is defined from a mixture of the three bases dA, dC or dG.
- the 'outer' 3 'nucleotide of the adapter overhang thus determines the specificity of the ligation.
- the ligation conditions are as described above. Each of the 256 ligation batches is used as a template after purification in a PCR (see (8b)).
- the cDNA synthesis primer (mixture of 12 anchor primers) which is completely substituted by 2'-O-methylated ribonucleotides is used as the 3 'primer. Due to the increased dissociation temperature of the modified bases of the oligonucleotide, a significantly higher (up to 40%, see for example LL Cummins 1995, Nucl.Acids Res., 23, 2019-2024) annealing temperature can be used in the PCR than with a unsubstituted primer.
- ⁇ rei 3 ' nucleotides correspond to the complete permutation of all four deoxyribonucleotides dA, dC, dG and dT.
- the primer permutations used here correspond to bases 3 - 4 or 2 - 4 of the adapter overhangs defined in the ligation approaches under (4a).
- a further 16 or 64 PCRs are carried out for each first PCR with the same 3 'primer and 16 5' oligonucleotides each with a length of 18-27 bases, which correspond to the 5 'primer of the first PCR, however, two or three 5 'nucleotides of complete permutation of all four deoxyribonucleotides dA, dC, dG and dT are extended.
- selective PCR primers are used, which can contain several artificially introduced fenipaires compared to the template DNA. These mismatches can be located anywhere on the oligomer, with 2 mismatches at positions -2 and -3 or 1 match at position -1 relative to the 3 ' end of the primer (position 0) preferred.
- the PCR profile is typically: 3 min, 95 S C followed by 20-40 cycles with 45 sec, 95 S C, 45 sec, 65 e C, 60 sec, 72 S C and a final extension for 60 sec at 72 ⁇ C, and is carried out with the aid of radioactive or fluorescent labeled PCR primers.
- the PCR is carried out as described under 8a), but using an unmodified cDNA synthesis primer (mixture of 12 anchor primers), which consists of standard deoxyribonucleotides.
- oligonucleotides with a length of 18-27 bases corresponding to the 3 ' end of the sense oligonucleotide of the second ligated adapter molecule (see (7)) are used as the 3' primer. - 19 -
- the PCR profile is typically: 3 min, 95 e C followed by 20-40 cycles with 45 sec, 95 e C, 45 sec, 65 e C, 60 sec, 72 S C and a final extension for 60 sec at 72 S C, and is carried out with the aid of radioactive or fluorescently labeled PCR primers.
- a 5 'primer with a length of 18-27 bases is inserted, which corresponds to the 3' end of the sense oligonucleotide of the ligated adapter molecule.
- the cDNA synthesis oligonucleotide is used as the 3 'primer.
- the PCR profile is typically: 3 min, 95 S C followed by 20-40 cycles with 45 sec, 95 Q C, 45 sec, 65 Q C, 60 sec, 72 Q C and a final extension for 60 sec at 72 e C, and is carried out in the presence of radioactively labeled nucleotides.
- the PCR fragments are typically analyzed on 6% polyacrylamide gels
- PAA gels with 7-8 M urea or by capillary electrophoresis.
- the results show that the selectivity for the amplification of the correct 5'-nucleotides of the cDNA for the 5'-primers of the TOGA method is not sufficient, in particular if the aim is to carry out a computer-assisted database analysis of the results .
- the error rate when using the 5 'primers according to the invention is 5 5%.
- the error rate can be further reduced to approximately 0% using the liagtion method described above. In view of this extremely low error rate, the establishment of an automated data analysis is also made possible.
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Application Number | Priority Date | Filing Date | Title |
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US09/622,247 US6670121B1 (en) | 1998-02-17 | 1999-02-16 | Methods for characterizing mRNA molecules |
AU28342/99A AU2834299A (en) | 1998-02-17 | 1999-02-16 | Methods for characterising mrna molecules |
EP99908907A EP1055003A1 (de) | 1998-02-17 | 1999-02-16 | VERFAHREN ZUR CHARAKTERISIERUNG VON mRNA-MOLEKÜLEN |
JP2000532550A JP3533373B2 (ja) | 1998-02-17 | 1999-02-16 | mRNA分子を特徴付けする方法 |
CA002322068A CA2322068A1 (en) | 1998-02-17 | 1999-02-16 | Methods for characterising mrna molecules |
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DE19806431A DE19806431C1 (de) | 1998-02-17 | 1998-02-17 | Neues Verfahren zur Identifikation und Charakterisierung von mRNA-Molekülen |
DE19806431.4 | 1998-02-17 |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002008461A2 (en) * | 2000-07-21 | 2002-01-31 | Global Genomics Ab | A METHOD AND AN ALGORITHM FOR mRNA EXPRESSION ANALYSIS |
GB2365124A (en) * | 2000-07-21 | 2002-02-13 | Karolinska Innovations Ab | Analysis and identification of transcribed genes, and fingerprinting |
WO2002036828A2 (en) * | 2000-11-01 | 2002-05-10 | Genomic Solutions Inc. | COMPOSITIONS AND SYSTEMS FOR IDENTIFYING AND COMPARING EXPRESSED GENES (mRNAs) IN EUKARYOTIC ORGANISMS |
WO2003064689A2 (en) * | 2002-01-29 | 2003-08-07 | Global Genomics Ab | Methods for identifying polyadenylation sites and genes thereof |
EP1347063A1 (de) * | 2002-03-20 | 2003-09-24 | Biofrontera Pharmaceuticals AG | Mehrfache Gene relevant für die Kennzeichnung, Diagnose und Manipulation des Schlaganfalls |
EP1434876A1 (de) * | 2001-09-11 | 2004-07-07 | The REGENTS OF THE UNIVERSITY OF COLORADO, A Body Corporate | Erstellen eines expressionsprofils im intakten menschlichen herz |
Families Citing this family (1)
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US7871571B2 (en) * | 2007-10-25 | 2011-01-18 | Parker John A | Biomolecule analyzing system |
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- 1999-02-16 WO PCT/EP1999/000992 patent/WO1999042610A1/de not_active Application Discontinuation
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Cited By (12)
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WO2002008461A2 (en) * | 2000-07-21 | 2002-01-31 | Global Genomics Ab | A METHOD AND AN ALGORITHM FOR mRNA EXPRESSION ANALYSIS |
GB2365124A (en) * | 2000-07-21 | 2002-02-13 | Karolinska Innovations Ab | Analysis and identification of transcribed genes, and fingerprinting |
GB2365124B (en) * | 2000-07-21 | 2002-05-01 | Karolinska Innovations Ab | Methods for analysis and identification of transcribed genes and fingerprinting |
WO2002008461A3 (en) * | 2000-07-21 | 2002-05-10 | Global Genomics Ab | A METHOD AND AN ALGORITHM FOR mRNA EXPRESSION ANALYSIS |
WO2002036828A2 (en) * | 2000-11-01 | 2002-05-10 | Genomic Solutions Inc. | COMPOSITIONS AND SYSTEMS FOR IDENTIFYING AND COMPARING EXPRESSED GENES (mRNAs) IN EUKARYOTIC ORGANISMS |
WO2002036828A3 (en) * | 2000-11-01 | 2003-02-27 | Genomic Solutions Inc | COMPOSITIONS AND SYSTEMS FOR IDENTIFYING AND COMPARING EXPRESSED GENES (mRNAs) IN EUKARYOTIC ORGANISMS |
US6955876B2 (en) | 2000-11-01 | 2005-10-18 | Kane Michael D | Compositions and systems for identifying and comparing expressed genes (mRNAs) in eukaryotic organisms |
EP1434876A1 (de) * | 2001-09-11 | 2004-07-07 | The REGENTS OF THE UNIVERSITY OF COLORADO, A Body Corporate | Erstellen eines expressionsprofils im intakten menschlichen herz |
EP1434876A4 (de) * | 2001-09-11 | 2005-05-25 | Univ Colorado Regents | Erstellen eines expressionsprofils im intakten menschlichen herz |
WO2003064689A2 (en) * | 2002-01-29 | 2003-08-07 | Global Genomics Ab | Methods for identifying polyadenylation sites and genes thereof |
WO2003064689A3 (en) * | 2002-01-29 | 2003-11-13 | Global Genomics Ab | Methods for identifying polyadenylation sites and genes thereof |
EP1347063A1 (de) * | 2002-03-20 | 2003-09-24 | Biofrontera Pharmaceuticals AG | Mehrfache Gene relevant für die Kennzeichnung, Diagnose und Manipulation des Schlaganfalls |
Also Published As
Publication number | Publication date |
---|---|
JP2002504348A (ja) | 2002-02-12 |
DE19806431C1 (de) | 1999-10-14 |
JP3533373B2 (ja) | 2004-05-31 |
CA2322068A1 (en) | 1999-08-26 |
EP1055003A1 (de) | 2000-11-29 |
US6670121B1 (en) | 2003-12-30 |
AU2834299A (en) | 1999-09-06 |
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