WO2002090371A2 - Verfahren zur präparativen herstellung von langen nukleinsäuren mittels pcr - Google Patents
Verfahren zur präparativen herstellung von langen nukleinsäuren mittels pcr Download PDFInfo
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- WO2002090371A2 WO2002090371A2 PCT/DE2002/001047 DE0201047W WO02090371A2 WO 2002090371 A2 WO2002090371 A2 WO 2002090371A2 DE 0201047 W DE0201047 W DE 0201047W WO 02090371 A2 WO02090371 A2 WO 02090371A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
- C12N15/68—Stabilisation of the vector
Definitions
- the invention relates to a method for the preparative production of long nucleic acids by means of PCR and various possible uses for such a method.
- the preparative scale refers to nucleic acid quantities obtained which are suitable for direct use in cell-free protein biosynthesis systems and / or in vitro transcription systems.
- Long nucleic acids are those nucleic acids which, in addition to a nucleic acid base sequence (of any length) coding for a protein, contain further sequences, in particular regulatory sequences, each with more than 50, even more than 70, nucleotides.
- Nucleic acids can be DNA or RNA, but also PNA.
- Proteins are used in high purity for biotechnological and medical applications, but especially in large quantities, i.e. on a mg and g scale. In the case of larger proteins, classic synthesis is hardly possible and at least uneconomical.
- RNA, mRNA can also be used directly. In this way, not only those proteins can be produced in a short time and with a comparatively moderate effort, which can also be genetically engineered, but even proteins can be produced which are, for example, cell toxic and consequently cannot be expressed at all with the usual genetic engineering cell systems.
- nucleic acid itself, which in turn is expensive using genetic engineering methods.
- regulatory sequences that are not naturally linked to a protein sequence, as well as other sequences, such as spacers, in order to improve the efficiency of protein synthesis.
- Expression PCR is an alternative to the genetic engineering of complete nucleic acids that can be used in cell-free protein biosynthesis.
- the efficient introduction of regulatory sequences (as well as other sequences that promote translation efficiency) into a nucleic acid to be produced plays a role in the context of
- Amplification plays a special role. Very long PCR primers are necessary for the introduction of such further sequences into a target nucleic acid. On the one hand, long primers are complex to manufacture and on the other hand increase the likelihood of generating inhomogeneous PCR products.
- nucleic acid From US-A-5, 571, 690 it is known to prepare a nucleic acid on a preparative scale by means of PCR, the nucleic acid to be amplified already containing all the required regulatory sequences. With the measures known in this respect, there is therefore no introduction of other, better regulatory sequences or replacement of the existing regulatory sequences by such other better ones Sequences possible.
- nucleic acid obtained from the amplification cannot be used directly in protein synthesis.
- a specific nucleic acid from a nucleic acid mixture cannot be specifically amplified, with simultaneous conversion of the target gene encoded by the specific nucleic acid for protein biosynthesis.
- the third stage is the overlap and extension response.
- the products from the first two stages are hybridized, filled into a double strand and finally amplified with further primers.
- an additional sequence is inserted via a primer in front of the promoter, which is supposed to bring about an improved transcription.
- transcription and translation take place in one cell-free system.
- the disadvantage here is that a total of four steps are necessary to obtain the desired RNA.
- 3 'sequences are missing, which are important for protein biosynthesis in prokaryotic systems.
- no Affmitatstagsequenzen or the like by means of which a purification of the protein obtained is facilitated. Due to the complexity of the method and the lack of 3 'sequences for prokaryotic systems, the method known in this respect could not be useful for prokaryotic systems or for an application to nuclear acid mixtures (cDNA or genome libraries).
- the invention is based on the technical problem of specifying a process for the production of long nucleic acids, in particular with protein sequences and with selected regulatory sequences, which works with little effort, gives high amounts of product nucleic acids, is suitable for prokayontic systems without additional effort and by means of whose amplification of defined nucleic acids from nucleic acid libraries is possible.
- the invention teaches a method for the preparative production of long nucleic acids by means of PCR and with the following hybridization steps: a) a nucleic acid base sequence is hybridized at the 3 'and 5' end with an adapter primer, b) the product from step a) is hybridized at the 3 'and 5' end with one extension primer each, which contains an extension sequence, with one from the nucleic acid base sequence at the 3 'and the 5' end of the Nucleic acid base sequence around the extended and amplified nucleic acid sequence is formed.
- a nucleic acid base sequence is a sequence which codes for a protein. In particular, this can be a gene, but also sequences from mtronless genomes.
- the extension sequences can in particular be sequences which comprise a regulatory sequence and / or sequences which contain a ⁇ bosomal binding sequence.
- the adapter primers are comparatively short. Part of an adapter sequence is specific for the nucleic acid base sequence, another part is constant and hybridizes to an extension sequence.
- extension sequences do not have to be used for different nucleic acid base sequences. Rather, only the comparatively short adapter primers have to be matched to a defined nucleic base sequence, while the extension sequences can be, so to speak, universal, ie the same or a few selected extension sequences can always be used for different nucleic acid base sequences.
- the extension sequences which are relatively complex to produce, can thus be used for a wide range of uses, and only the adapter sequences have to be produced for a specific nucleic acid base sequence. However, this is not very expensive since the adapter sequences can be comparatively short.
- a particular advantage of the method according to the invention is that it is a generally applicable method for any coding sequences.
- the hybridization with a primer at the 3 'and 5' end relates in particular to double-stranded nucleic acids, the hybridization of the pri ers taking place at the 5 end of the sense and antisense strand. In relation to the single strand, the hybridization with the various primers described above and below takes place at the respective 5 'end.
- a further development of the invention is of independent importance, wherein the product from stage b) in stage c) can be hybridized with an amplification primer at the 3 'end and at the 5' end, an amplified nuclear acid end sequence being formed.
- the amplification primers are comparatively short and can be used universally and are therefore readily available.
- the Amplification primers can also have further (shorter) sequences added at the ends, which further increase the translation efficiency.
- variations and modifications at the ends of the nucleic acids can also be introduced with little effort. This is particularly advantageous because it does not require different extension primers to be produced for variations and modifications, which in turn would be troublesome to an extent.
- An example of a variation or modification is the incorporation of a biotin residue, coupled at the 5 'end of the amplification primer.
- a nucleic acid end sequence stabilized against exonuclease degradation is obtained, which increases the half-life in an in vitro protein biosynthesis system by a multiple, typically more than 5 times, for example, compared to an unstabilized nucleic acid sequence of approx. 15 min. to about 2 h.
- Stabilities are achieved which are comparable to those of circular plasmids and can therefore replace them to the same extent.
- An alternative is stabilization using digoxygenin, which binds anti-digoxygenin antibodies.
- the stabilizing group can in particular be set up at both ends of the nucleic acid end sequence.
- Another example is a modification with an affinity tag or a sequence coding therefor or an anchor group or a sequence coding therefor.
- An anchor group allows immobilization by binding the Anchor group on a solid surface with matched binding sites.
- the anchor group can be attached to the nucleic acid itself, but a sequence coding therefor can also be provided.
- the adapter primers typically contain ⁇ 70, in particular 20-60, nucleotides.
- the extension primers typically contain> 70, also 90 and more, nucleotides.
- the amplification primers in turn typically contain ⁇ 70, mostly ⁇ 30, nucleotides, typically> 9 nucleotides. Only the adapter primers have to be specifically adapted to a defined nucleic acid base sequence, which is associated with little effort in the light of the relatively short sequences.
- Steps a), b) and optionally step c) are advantageously carried out in a PCR solution containing the nucleic acid base sequence, the adapter primers, the extension primers and optionally the amplification primers. It is then a one-step PCR with a total of 6 primers, two adapter sequences, two extension sequences and two amplification sequences. It is sufficient to use the adapter primer and extension primer in low concentrations and, in this respect, only to produce a small amount of intermediate product. The intermediate product also does not need to be homogeneous, which means that complex optimizations can be dispensed with. Because of the shortness of the amplification primers, no optimizations are necessary even for the amplification to the high amount of nucleic acid end sequence.
- Steps a) and b) are carried out in a process step A) in a pre-PCR solution containing the nucleic acid base sequence, the adapter primer and the extension primer for a defined first number of cycles, and step c) is carried out in a process step B) in a main PCR solution containing the PCR product from stage A) and the amplification primers carried out for a defined second number of cycles.
- Step A) can be carried out in a reaction volume which is 1/2 to 1/10 of the reaction volume of step B). In step A), a higher concentration of intermediate product arises due to the lower volume or significantly less nucleic acid base sequence can be used.
- the adapter primers and the extension primers are again greatly diluted, with the result that the probability of incorporating variations and / or modifications into the nucleic acid end sequences via the amplification primers is increased ,
- the procedure can be such that the PCR in a reaction volume of 10 to 100 ⁇ l, preferably 20 to 40 ⁇ l, with 0.01 to 100 pg, preferably 1 to 50 pg, nucleic acid base sequence, 0.05 to 10 ⁇ M, preferably 0.1 to 5 ⁇ M, adapter primer and 0.005 to 0.5 ⁇ M, preferably 0.001 to 0.1 ⁇ M, extension primer is carried out, with 0.01 to 10 ⁇ M, preferably 0.1 to 10, after a defined number of initial cycles uM, Amplification primers are added, and the amplified nucleic acid end sequence is produced by means of a defined number of subsequent cycles.
- Step A reaction volume ⁇ 10 ⁇ l; 0.001 to 5 pg, preferably 0.01 to 1 pg, nucleic acid base sequence; 0.05 to 10 uM, preferably 0.1 to 5 uM, adapter primer and 0.05 to 10 uM, preferably 0.1 to 5 uM, extension primer; first number of cycles 10 to 30, preferably 15 to 25,
- the invention further teaches the use of a method according to the invention for the production of nucleic acids for cell-free in vitro protein biosynthesis, in particular in prokaryotic systems, preferably in a translation system from Escheria coli D10.
- a method according to the invention can advantageously be used for the selective amplification of a defined nucleic acid base sequence from a nucleic acid library. This makes it possible to characterize gene sequences, the gene sequence being used as the nucleic acid base sequence and the protein obtained being analyzed with regard to structure and / or function.
- the background to this aspect of the invention is that for many genes the sequences are known, but not the structure and function of the protein encoded thereby.
- elements of a gene library, for which only the sequence as such is known can be examined for their function in an organism. The study of the structure and function of the protein obtained follows the usual working methods of biochemistry.
- the method according to the invention can be used to obtain nucleic acids which contain a nucleic acid base sequence coding for a protein and a ⁇ bosomal binding sequence and optionally one or more sequences from the group consisting of "promoter sequence, transcription terminator sequence, expression enhancer sequence, stabilization sequence and affinity tag sequence" ,
- An affinity tag sequence codes for a structure which has a high affinity for (usually immobilized) binding sites in separation systems for purification. This enables easy and high-affinity separation of proteins that do not contain the affinity tag.
- An example of this is Strep-tag II, a peptide structure consisting of 8 amino acid residues with affinity for StrepTactm.
- a stabilization sequence codes for a structure which, either by itself or after binding with an binding molecule specific for the structure, brings about stabilization against degradation, in particular by nucleases.
- a nucleic acid (end) sequence can also be stabilized by incorporating a biotin group at one end, preferably at both ends, which can be reacted with streptavid. This installation can be carried out by using primers carrying biotin, in particular amplification primers.
- An expression enhancer sequence increases the translation efficiency compared to a nucleic acid without an expression enhancer sequence. This can be, for example, (non-translated) spacers.
- a transcription terminator sequence terminates RNA synthesis. An example of this is the T7 Phage Gen 10 transcription terminator. Transcription terminator sequences can also stabilize against degradation by 3 'exonucleases. Advantageous relative arrangements of the above sequence elements to one another can be generalized from the following exemplary embodiments.
- PCR The PCR was performed in a quantified in Examples reaction volume with 10 mM Tris-HCl (pH 8.85 at 20 ° C), 25 mM KCl, 5 mM (NH 4) 2 S0 4, 2 mM MgS0 4, 0 25 mM of each dNTP, 3 U Pwo DNA polymerase (Röche) and the amount of nucleic acid base sequence given in the examples were carried out. The cycles were for 0.5 min. at 94 ° C, 1 min. at 55 ° C and 1 min. performed at 72 ° C.
- the reactions were carried out at 37 ° C., the course being followed by taking 5 ⁇ l aliquots of the reaction mixture at subsequent times and estimating the incorporation of [ 14 C] Leu by means of TCA precipitation. A further 10 ⁇ l aliquots were removed for the analysis of the synthesized protein using SDS-PAGE, followed by autoradiography in a phosphoimager system (Molecular Dynamics).
- Plasmid construction A high copy derivative of the plasmid pET BH-FABP
- pHMFA bovine heart fatty acid binding protein
- the plasmid pHMFA served as a template for the construction of nucleic acids with different sequence regions upstream of the promoter.
- the constructs (see examples) FA1, FA2 and FA4 with 0, 5 and 249 base pairs upstream of the promoter were primed with P1, Cl and P2 and generated with the downstream primer P3.
- the construct FA3 with a sequence region of 15 base pairs upstream of the promoter was obtained by digestion of FA4 with the endonuclease Bgl II.
- the control plasmid pHMFA (EcoRV) with a sequence range of 3040 base pairs was obtained by digesting the plasmid with EcoRV. All products were purified by agarose gel electrophoresis, followed by gel extraction using the "High Pure PCR Product Purification Kit".
- Radioactive labeled nucleic acids were synthesized according to the above conditions, but in the presence of 0.167 ⁇ Ci / ⁇ l [ ⁇ - 35 S] dCTP.
- the labeled nucleic acids were used in a coupled transcription / translation, reaction volume 400 ⁇ l. 30 ul aliquots were taken at successive times. After the addition of 15 ⁇ g ribonuclease A (DNAse-free, Röche), these were kept for 15 min. incubated at 37 ° C. Another incubation for 30 min. at 37 ° C., 0.5% SDS, 20 mM EDTA and 500 ⁇ g / ml Proteinase K (Gibco BRL) were added in a total reaction volume of 60 ⁇ l.
- Remaining PCR products were further purified using ethanol precipitation and then subjected to denaturing electrophoresis (5.3% polyacrylamide, 7 M urea, 0.1% SDS, TBE). The dried gel was run through a phosphoimager system (Molecular Dynamics) to quantify the radioactivity.
- Example 1 PCR with four primers
- FIG. 2 shows a schematic representation of a one-step PCR according to the invention with four primers.
- the nucleic acid base sequence coding for a protein which contains the complete coding sequence for H-FABP (homogeneous and functionally active fatty acid binding protein from bovine heart), obtained as a 548 bp restriction fragment from pHMFA by digestion using the endonucleases Ncol and BamHI, (and a 150 bp sequence at the 3 'end, which is neither translated, nor is complementary to an adapter primer or extension primer).
- the two adapter primers A and B are hybridized to them, which comprise ends homologous to the ends of the nucleic acid base sequence.
- the adapter primer A also contains a ribosomal binding sequence.
- the extension primers C and D are hybridized to the outer ends of the adapter primers A and B.
- the extension primer C comprises the T7 gene 10 leader sequence including the T7 transcription promoter and optionally upstream a sequence of, for example, 5 nucleotides.
- the extension primer D comprises the T7 gene 10 terminator sequence.
- Example 2 Efficiency of H-FABP synthesis depending on the sequence region upstream of the promoter.
- FIG. 3 shows that all sequence regions, except 0 (lower curve), increase the protein synthesis. 5 base pairs are sufficient.
- Example 3 Improvement of H-FABP synthesis by phage T7 gene 10 transcription terminator / 5 'leader sequence phage T7 gene 10.
- FIG. 4 shows that the phage T7 gene 10 transcription terminator can improve the synthesis by at least a factor of 2.8.
- the triangles stand for FA ⁇ t and the squares for FAt (see also Fig. 2).
- Example 4 Influence of the position of the transcription terminator sequence.
- the products FAst and FAast were generated to investigate the influence of the position of the terminator sequence. Both are identical to FAt and FAat, except that there is a 22 bp spacer sequence between the Stop codon and the terminator was introduced using different primers. 5 that the spacer sequence causes an approximately 2-fold increase in expression.
- Example 5 PCR from a complex DNA mixture.
- a PCR for FAst was carried out in accordance with the above descriptions with the following exceptions: the nucleic acid base sequence was carried out in concentrations of 0.16 to 20 pg / 50 ⁇ l reactor volume and the reactions were carried out with 0.83 ⁇ g chromosomal DNA from Escherichia coli, sonicated for 5 min., supplemented. It was found that neither the quality nor the quantity of the PCR product was affected by the presence of a 5 million fold excess of competitive DNA.
- a reaction mixture with 10 ⁇ g of the radiolabelled FAast was subjected to affinity purification. About 81% of the applied material was obtained from the column and 67% could be used as a pure product m are obtained from the elution fractions (calculated from TCA precipitation of the fractions of the affection column).
- Example 7 Activity of the PCR product.
- Example 8 Stability of the PCR product.
- the decrease in the PCR product FAast was measured.
- the radio-labeled product was used for this.
- aliquots of the reaction mixture were removed and examined using denaturing polyacrylamide gel electrophoresis.
- the amount of the remaining PCR product was quantified by scanning the radioactivity of the gel and compared with the time course of the protein synthesis, measured by scanning the radioactivity of H-FABP in the gel after separation of the reaction mixtures using SDS-PAGE.
- the results are in the Figure 6 shown. It can be seen that the half-life of the PCR product is approx. 100 min. is what corresponds to the entry of the H-FABP synthesis into a plateau.
- Example 9 Optimized conditions for a PCR with four primers.
- Table I summarizes optimized conditions for a PCR with four primers in a reaction volume of 25 ⁇ l.
- Example 10 PCR with six primers.
- BIOF is a biotin-labeled forward primer and BIOR is a biotin-labeled reverse primer.
- FIG. 7 The structure is shown in FIG. 7.
- Amplification primers were run.
- the concentration of extension primer could be reduced to 0.025 ⁇ M by using the amplification primer, a factor of approx. 1/20, but with improved homogeneity and yield of PCR product.
- Example 11 PCR with six primers in two stages.
- the materials are used as stated above.
- a pre-PCR is carried out in a reaction volume of 5 ⁇ l with 0.1 pg nucleic acid base sequence, namely with 0.3 ⁇ M adapter primer and 0.5 ⁇ M extension primer over 20 Cycles.
- the reaction solution thus obtained is diluted to 25 ⁇ l with a PCR batch volume.
- amplification primer is added to a final concentration of 0.5 ⁇ M.
- amplification is carried out for a further 30 cycles.
- Example 12 Stabilization of a nucleic acid with biotin.
- a nucleic acid was produced using the primers BIOF and BIOR by means of PCR with 6 primers, as described above, and its degradation as a function of time and the improvement in protein synthesis were investigated. This is shown in Figures 7 and 8. It can be seen that with biotin, especially after conversion with streptavidin, stability is considerably improved. This also leads to an up to 20% higher protein synthesis.
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Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
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DE50209080T DE50209080D1 (de) | 2001-03-16 | 2002-03-18 | Verfahren zur präparativen herstellung von langen nukleinsäuren mittels pcr |
AU2002311070A AU2002311070A1 (en) | 2001-03-16 | 2002-03-18 | Method for preparative production of long nucleic acids by pcr |
US10/472,003 US20060099577A1 (en) | 2001-03-16 | 2002-03-18 | Method for preparative production of long nucleic acids by pcr |
EP02735010A EP1368500B1 (de) | 2001-03-16 | 2002-03-18 | Verfahren zur präparativen herstellung von langen nukleinsäuren mittels pcr |
JP2002587448A JP4316891B2 (ja) | 2001-03-16 | 2002-03-18 | Pcrを手段とする長い核酸の調製方法 |
US12/365,702 US20110212452A1 (en) | 2001-03-16 | 2009-02-04 | Method for preparative production of long nucleic acids by pcr |
US13/630,643 US20130115605A1 (en) | 2001-03-16 | 2012-09-28 | Method for preparative production of long nucleic acids by pcr |
US14/605,513 US20150368686A1 (en) | 2001-03-16 | 2015-01-26 | Method for preparative production of long nucleic acids by pcr |
US15/388,164 US20170327859A1 (en) | 2001-03-16 | 2016-12-22 | Method for preparative production of long nucleic acids by pcr |
US16/251,184 US20190382818A1 (en) | 2001-03-16 | 2019-01-18 | Method for preparative production of long nucleic acids by pcr |
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DE10113265.4A DE10113265B4 (de) | 2001-03-16 | 2001-03-16 | Verwendung einer stabilisierten Nukleinsäure zur Herstellung eines Proteins |
DE10113265.4 | 2001-03-16 |
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US10/472,003 A-371-Of-International US20060099577A1 (en) | 2001-03-16 | 2002-03-18 | Method for preparative production of long nucleic acids by pcr |
US12/365,702 Continuation US20110212452A1 (en) | 2001-03-16 | 2009-02-04 | Method for preparative production of long nucleic acids by pcr |
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US (6) | US20060099577A1 (de) |
EP (1) | EP1368500B1 (de) |
JP (1) | JP4316891B2 (de) |
AT (1) | ATE349552T1 (de) |
AU (1) | AU2002311070A1 (de) |
DE (2) | DE10113265B4 (de) |
WO (1) | WO2002090371A2 (de) |
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WO2013122826A1 (en) * | 2012-02-14 | 2013-08-22 | Gnubio, Inc. | Cascaded addition of target specific universal adapters to nucleic acids |
EP3475375B1 (de) * | 2016-06-22 | 2023-11-15 | CMC Materials, Inc. | Polierzusammensetzung mit einem aminhaltigen tensid |
US10822647B2 (en) * | 2016-07-12 | 2020-11-03 | Biodynamics S.R.L. | Methods for using long ssDNA polynucleotides as primers (superprimers) in PCR assays |
Citations (1)
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WO1992007949A1 (en) * | 1990-11-05 | 1992-05-14 | The United States Of America, As Represented By The Secretary Of The Army | Method for the in vitro production of protein from a dna sequence without cloning |
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DE4106473C2 (de) * | 1991-03-01 | 1994-03-10 | Boehringer Mannheim Gmbh | Verfahren zur Herstellung in vitro replizierbarer Nukleinsäuren |
US5571690A (en) * | 1991-05-08 | 1996-11-05 | The University Of Virginia Patents Foundation | Method for the cell-free synthesis of proteins |
US5834247A (en) * | 1992-12-09 | 1998-11-10 | New England Biolabs, Inc. | Modified proteins comprising controllable intervening protein sequences or their elements methods of producing same and methods for purification of a target protein comprised by a modified protein |
DE19518505A1 (de) | 1995-05-19 | 1996-11-21 | Max Planck Gesellschaft | Verfahren zur Genexpressionsanalyse |
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2002
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- 2002-03-18 WO PCT/DE2002/001047 patent/WO2002090371A2/de active IP Right Grant
- 2002-03-18 JP JP2002587448A patent/JP4316891B2/ja not_active Expired - Lifetime
- 2002-03-18 EP EP02735010A patent/EP1368500B1/de not_active Expired - Lifetime
- 2002-03-18 AT AT02735010T patent/ATE349552T1/de active
- 2002-03-18 US US10/472,003 patent/US20060099577A1/en not_active Abandoned
- 2002-03-18 DE DE50209080T patent/DE50209080D1/de not_active Expired - Lifetime
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2009
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2012
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2015
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2016
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2019
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Also Published As
Publication number | Publication date |
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US20190382818A1 (en) | 2019-12-19 |
DE10113265A1 (de) | 2002-10-02 |
US20060099577A1 (en) | 2006-05-11 |
DE50209080D1 (de) | 2007-02-08 |
US20170327859A1 (en) | 2017-11-16 |
US20150368686A1 (en) | 2015-12-24 |
EP1368500B1 (de) | 2006-12-27 |
AU2002311070A1 (en) | 2002-11-18 |
US20130115605A1 (en) | 2013-05-09 |
DE10113265B4 (de) | 2018-03-08 |
JP2004524854A (ja) | 2004-08-19 |
WO2002090371A3 (de) | 2003-07-24 |
ATE349552T1 (de) | 2007-01-15 |
JP4316891B2 (ja) | 2009-08-19 |
US20110212452A1 (en) | 2011-09-01 |
EP1368500A2 (de) | 2003-12-10 |
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