EP1432797A2 - Procedes de manipulation d'adn - Google Patents

Procedes de manipulation d'adn

Info

Publication number
EP1432797A2
EP1432797A2 EP02779456A EP02779456A EP1432797A2 EP 1432797 A2 EP1432797 A2 EP 1432797A2 EP 02779456 A EP02779456 A EP 02779456A EP 02779456 A EP02779456 A EP 02779456A EP 1432797 A2 EP1432797 A2 EP 1432797A2
Authority
EP
European Patent Office
Prior art keywords
vector
complementary
nucleic acid
oligonucleotides
stranded
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02779456A
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German (de)
English (en)
Inventor
Thomas Rudel
Nikolaus Machuy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Max Planck Gesellschaft zur Foerderung der Wissenschaften eV filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Publication of EP1432797A2 publication Critical patent/EP1432797A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N15/66—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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids

Definitions

  • the present invention relates to methods and reagent kits for DNA manipulation, in particular for site-specific mutagenesis or for cloning.
  • nucleic acids For the investigation of structure-function relationships, e.g. the kinase function depending on certain amino acids or the promoter function of DNA segments depending on their sequence, it is often necessary to carry out a targeted mutagenesis of nucleic acids.
  • Hutchison et al. (Hutchison et al., 1 978) describe a site-directed mutagenesis in which single-stranded DNA is hybridized with an oligonucleotide which, except for the base to be mutagenized, is complementary to the template DNA. After extending the oligonucleotide, i.e. creating a double strand with a DNA polymerase, closing the synthesized strand with a ligase and transforming it into E. coli, should actually contain 50% of the clones mutated DNA. In practice, however, this rate is significantly lower (Kramer et al., 1 984).
  • mutagenesis is carried out similarly to the method described above, with the difference that, together with the mutagenesis oligonucleotide, two more
  • Hybridize primers that activate the Amp R gene and inactivate the Tet R gene (Kleina et al., 1990; Normanly et al., 1 990).
  • the Transformer TM Site-Directed Mutagenesis Kit from Clontech uses a second oligonucleotide to be hybridized, which is used to destroy a restriction site that is unique in the starting construct (Deng and Nickoloff, 1 992; Zhü, 1 996). After hybridization and oligonucleotide extension, only the non-mutated plasmids are cut by digestion with this restriction enzyme, which plasmids can therefore no longer be transformed. This method also uses repair-deficient strains. In addition, two successive transformations are necessary, which significantly increases the duration of the mutagenesis.
  • the DNA section to be mutated is amplified in two separate PCRs in such a way that the products overlap at the mutation site by approximately 20 bases and already contain the mutated sequence (Lott Tips, F. and Zorbas, 1 998).
  • the two products are mixed and in a third PCR with oligonucleotides that allow cloning into the desired vector, the entire DNA is amplified.
  • the disadvantage of this method is that three PCRs are necessary.
  • Another mutagenesis method starts with two PCRs (Cooney, 1 998).
  • the two products overlap at the mutation site, but not 20 bases, but only so many that complementary ⁇ 'overhangs are produced by a T 4 DNA polymerase trimming reaction.
  • the outer ends of the PCR products are treated with restriction enzymes. Both products and the vector are mixed for ligation.
  • the disadvantages of this method are both the need to take into account restriction sites within the fragments and the ligation of three DNA fragments.
  • a first aspect of the present application is the subject of claim 1 and relates to a method for mutagenizing
  • Nucleic acids in particular double-stranded DNA fragments, comprising the steps:
  • nucleic acid template to be mutagenized, for example a plasmid vector
  • oligonucleotides which (i) each hybridize with a strand of the nucleic acid template and (ii) in the region of its 5 'ends a short, preferably 2-4
  • Base pairs have a long, complementary section
  • target sequence which is preferably in the range of 5'-
  • oligonucleotides are produced which, for example, contain the mutated DNA sequences at the ⁇ 'ends and which preferably have an overlap of 2-3 base pairs in length (here AA or TT).
  • the length of the oligonucleotides is chosen so that they can bind to the template with sufficient efficiency under suitable hybridization conditions.
  • the length of the oligonucleotides is usually, for example, 1 to 50 bases.
  • the rate of mutagenesis can be additionally increased by means of p? l digestion, whereby only methylated DNA is cut.
  • a negative control a ligation approach without
  • the approach without ligase cannot deliver transformants.
  • the mutation can be checked after transformation of the mutagenized
  • PCR control is carried out using an oligonucleotide that only leads to a product with mutated DNA. The first ten with this
  • the mutagenesis method according to the invention in particular carries out DNA manipulations, selected from the group: point mutations, an exchange or substitution of one or more individual nucleotides taking place; Insertions, in which one or more individual nucleotides are additionally inserted into the sequence; Inversions, one or more sequences of at least two bases, e.g. two, three or four bases, can be turned over; Deletions, where one or more individual bases are removed; and any combinations of the mutagenesis options mentioned, e.g. two or more combinations of point mutation, insertion, inversion and / or deletion in a single step or insertion of several point mutations with a maximum distance, corresponding to the length of the oligonucleotides used, e.g. 50 bases in one step.
  • the advantage of the method according to the invention over other mutagenesis methods is the extremely high mutagenesis rate of 95%.
  • the Generation of stepped ends by trimming, for example by means of a T 4 DNA polymerase reaction means that only accurately trimmed products can religion. In contrast to this, reading frame shifts due to an additional base, for example an adenine, can occur during the religation of untrimmed amplification products. In contrast to many commercially available systems, one is not dependent on the existence of singular restriction interfaces as well as certain antibiotic resistance.
  • Another advantage of the process is its speed. On the first day, PCR, T 4 DNA polymerase reaction, ligation, Dpnl digestion and transformation can be carried out. On the second day the colonies are analyzed by PCR and cultures for plasmid preparation are set up. The mutated DNA can be isolated and used in experiments on the third day.
  • this first aspect of the invention also relates to a reagent kit, in particular for carrying out the mutagenesis method described above, comprising: (a) means for nucleic acid amplification, (b) means for trimming double-stranded nucleic acids to produce stepped ends, that are complementary to each other,
  • the components of the reagent kit can be obtained together or from different sources.
  • the kit can also contain customary buffer and auxiliary reagents for carrying out the reactions as well as a written description of the process.
  • a second aspect of the invention relates to a new cloning method, with which the difficulties associated with the formation of multimers in both cloning doubles are more difficult Nucleic acid fragments, for example oligonucleotides with a length of preferably up to 200 bp, particularly preferably from 1 5-100 bp, or also longer PCR fragments, can be removed.
  • Nucleic acid fragments for example oligonucleotides with a length of preferably up to 200 bp, particularly preferably from 1 5-100 bp, or also longer PCR fragments, can be removed.
  • two complementary oligonucleotides are usually used which, after hybridization, have complementary overhangs to a vector opened with corresponding restriction enzymes and can be ligated therein.
  • the problem often arises that the double-stranded oligonucleotides form multimers and therefore either cannot be cloned at all or integrate them into the vector as multimers. This problem can be avoided by the method according to the invention.
  • the vector is opened with two different restriction enzymes and one or two nucleotides are filled in at the DNA ends using the Klenow polymerase. This creates different stepped ends that are not complementary to each other and not complementary to themselves ( Figure 2). The ends of the oligonucleotides are selected so that they are complementary to the filled-in vector ends.
  • the part into which the fragment to be cloned is to be inserted is amplified, for example by means of PCR. Then, by trimming, for example using T 4 DNA polymerase, using certain stop nucleotides, stepped ends are produced which are not complementary to themselves and not complementary to one another. To achieve this, the design of the oligonucleotides suitable bases are provided at the 5 'ends.
  • Variant 2 allows the insertion of double-stranded oligonucleotides at any position in a vector, for example a plasmid.
  • oligonucleotides To produce double-stranded oligonucleotides, equivalent amounts of the single-stranded oligonucleotides are hybridized with one another under suitable conditions, for example by mixing in the presence of MgCl 2 , heating and slow cooling from 95 ° C. to room temperature.
  • the ends of the hybridized oligonucleotides preferably have 5 'overhangs which are complementary to the 5' overhangs of the prepared vectors. Since the 5 'overhangs of the double-stranded DNA thus formed are not complementary to one another and to themselves, no multimers can be formed. This leads to an enormous increase in the efficiency of the cloning of oligonucleotides.
  • the incorporation of the oligonucleotides can be checked by PCR or / and restriction digestion and sequencing.
  • Another aspect of the invention is the subject of claim 8 and relates to a method for cloning nucleic acid fragments into a vector according to variant 1 comprising the steps:
  • this embodiment relates to a reagent kit, in particular for carrying out the cloning process, comprising:
  • Nucleoside triphosphates the ends of which are not complementary to one another and not complementary to themselves, and
  • (c) means for ligating nucleic acids.
  • the reagent kit can also contain customary buffer and auxiliary reagents as well as a written description of the process.
  • Yet another aspect of the invention is the subject of claim 1 3 and relates to a method for cloning nucleic acid fragments into a vector according to variant 2, comprising the steps:
  • Preferred embodiments of this method are the subject of claims 1 4 to 1 5.
  • this embodiment relates to a
  • Reagent kit especially for performing the aforementioned
  • (c) means for ligating nucleic acids.
  • the reagent kit can also contain conventional buffer and auxiliary reagents and a description of the process.
  • Another new possibility in connection with open vectors or plasmid parts produced according to variant 2 is to insert amplification products trimmed with T 4 DNA polymerase, for example PCR products.
  • cloning with this method is independent of restriction sites.
  • the stepped ends of the PCR products to be inserted must be complementary to the vector ends. This makes it possible to insert certain DNA sections very precisely and without additional, sometimes disturbing bases. This is particularly helpful if the fusion proteins are composed, for example, of a signal peptide and the protein to be examined. Furthermore, functional domains can be exchanged between different proteins at will.
  • Yet another aspect of the invention is the subject of claim 1 7 and relates to a method for cloning nucleic acid fragments into a vector, comprising the steps:
  • this embodiment relates to a reagent kit, in particular for carrying out the aforementioned cloning process, comprising:
  • (c) means for trimming double stranded nucleic acids to produce stepped ends which are not complementary to each other and not complementary to themselves, and
  • (d) means for ligating nucleic acids.
  • the reagent kit can also contain conventional buffer and auxiliary reagents and a description of the process.
  • the preferred annealing temperature of the oligonucleotides is specified and determined according to the 4 + 2 rule (2 ° C. for each adenine and thymine nucleotide; 4 ° C. for each guanine and cytosine nucleotide) using the base sequence of the oligonucleotide.
  • the difference in the annealing temperature of the two oligonucleotides should preferably be at most 2 ° C.
  • the base thymidine should preferably be avoided at the 3 'end of the oligonucleotides.
  • Ends of the amplification products are said to be phosphorylated.
  • Either the desired mutation is specified, e.g. on
  • Amino acid exchange, and the bases to be changed are determined by the algorithm, or only the desired mutation, e.g. a base exchange is specified. In both cases, additional silent mutations can be inserted.
  • the preparation of the vector by restriction digestion and subsequent filling reaction e.g. by the Klenow enzyme, the following algorithm applies:
  • Possible restriction interfaces are specified.
  • the program is designed to determine an interface combination with a nucleotide specification for the Klenow reaction. Furthermore, the 5 ' overhangs of the double-stranded oligonucleotides to be inserted, the must be complementary to the vector ends, but must not be complementary to themselves.
  • the exact insertion point is specified. This can also include a deletion.
  • T 4 - DNA polymerase reaction the ends of the PCR products must not be complementary to each other or to themselves.
  • the oligonucleotides used for the PCR must be phosphorylated at their 5 'ends.
  • a sequence should be sought starting from the specified insertion point, which will generate the desired properties both in the Vector as well as allowed at the ends of the oligonucleotides to be inserted.
  • the ends of the oligonucleotides for PCR or insertion are lengthened or shortened accordingly. It is also possible to introduce the bases necessary for the cloning via the oligonucleotide.
  • the length of the oligonucleotides to be inserted is arbitrary and is only limited by the given possibilities of oligonucleotide synthesis.
  • the stepped ends of the double-stranded oligonucleotides to be inserted during the dimerization must be complementary to the vector ends. On the other hand, they must not be complementary to themselves.
  • Figure 1 shows an example of the implementation of the mutagenesis method according to the invention.
  • Figure 1A shows the mutagenesis to be carried out (replacement of G / C with A / T, combined with an amino acid exchange Asp according to Asn).
  • Figure 1 B shows the schematic implementation of the process.
  • the starting construct to be mutagenized is amplified, for example by PCR.
  • the 5 'ends of the oligonucleotides used as primers contain the mutated sequence.
  • a T 4 DNA polymerase trim reaction produces staged complementary ends that can be ligated together.
  • the positive clones can be identified by suitable measures, such as PCR and / or sequencing.
  • Figure 2 shows the cloning of oligonucleotides according to variant 1 of the cloning method according to the invention.
  • the circular vector is for this purpose with 2 restriction enzymes, e.g. Nhel and BamHI, which do not result in complementary stepped ends, are cut.
  • the stepped ends are partially filled by treatment with Klenow enzymes.
  • the sequence of the oligonucleotides or amplification products to be cloned into the vector is defined such that they have 5 'overhangs which are complementary to the vector as a double-stranded fragment and can be ligated with the vector.
  • the conditions for the PCR were chosen according to the table below. Since the amplification of an entire plasmid leads to long PCR products, the reaction batches were supplemented with 5% by volume glycerol and 2-5% by volume DMSO. In addition, both the amount of template DNA ( ⁇ 10 ng) and the number of PCR cycles ( ⁇ 25) were kept as small as possible.
  • the cloning vectors were opened at the appropriate sites with the aid of restriction enzymes. Since not all enzymes cut in the same buffer and some enzymes work very poorly near the DNA ends, the following conditions were met : Conditions for restriction digestion after the choice of the enzymes. The conditions for cutting with Hin ⁇ ⁇ and Bam ⁇ are given below. First about 7 ⁇ g of the vector were digested for one hour with 10 U Hin ⁇ ⁇ in 30 ⁇ l 1 x buffer A (Röche) in an incubator at 37 ° C. After adding another 5 U Hin ⁇ ⁇ another hour was cut.
  • the buffer was changed either by adding the appropriate buffer and salt solutions or, as in this case, by
  • Buffer for the second enzyme In the example described here, the Vector digested in 1 x buffer B (Röche) and 10 U Bam ⁇ ⁇ in 30 ⁇ l final volume for one hour and after renewed enzyme addition (5 U) overnight. The next morning 5 U Bam ⁇ were added and incubated again for one hour. The restriction enzymes were then denatured at 65 ° C. for 10 min.
  • the DNA was then run through Centrifugation for 1 5 min at 1 3,000 rpm and 4 ° C, washed with 70% ethanol and dried at 37 ° C.
  • the prepared vector was resuspended in 30 ⁇ l H 2 O and the concentration was estimated by agarose gel electrophoresis.
  • the PCR approach below was pipetted and the PCR was carried out according to the program indicated. Since the amplification of almost the entire plasmid leads to long PCR products, the reaction batches were supplemented with 5% by volume glycerol and 2-5% by volume DMSO. In addition, both the amount of template DNA ( ⁇ 10 ng) and the number of PCR cycles ( ⁇ 25) were kept as small as possible.
  • the Qiagen PCR Purification Kit was used to purify the PCR products.
  • the PCR products were mixed with 5 times the volume of buffer PB and bound to a mini column by centrifugation. After washing with PE buffer, the DNA was eluted with 40 ⁇ l H 2 O.
  • the samples were denatured for 15 minutes at 75 ° C. and, as described for the vector preparation, purified and analyzed.
  • oligonucleotides For the dimerization of the oligonucleotides, 10 ⁇ g of primer in a 50 mM Tris-HCl buffer (pH 7.5) were mixed with 1.75 mM MgCl 2 in a final volume of 100 ⁇ l and in a PCR device at 95 ° C. denatured. The hybridization was carried out by slowly cooling the heating block to room temperature.
  • restriction enzymes the interface of which is not in the recognition sequence
  • the most serious advantage is the possibility of directed cloning with only one restriction enzyme. Since the efficiency of a restriction is much higher than that of a double digest, the cloning rate increases enormously. In addition, no linker fragment falls out of the vector, which therefore no longer has to be separated.
  • the recognition sequence for Xcm ⁇ used here nine Bases at the interface can be chosen freely. However, one could also use any other restriction enzyme which, in addition to a specific recognition sequence, allows the free choice of bases. Examples are Xmn ⁇ , Van91 ⁇ , Sfi ⁇ , Mwo ⁇ or others. This enables the user to insert bases which allow the use of oligonucleotides already present in the laboratory. The high efficiency achieved with this cloning technique makes it suitable for automated cloning of DNA.
  • the Xc l interface was inserted into the vector pDLO 1 2 via a Di- / TriSec mutagenesis with the oligonucleotides pDLO 1 x-5'-Xc / 77l (5'-Phos-TTATCGATGGATCCAGACATGATAAGATACATTGA-3 ') and pDLO 1 - 3'- ca7? L (5'-Phos-ATAAGGTGGCAGGTCGGATCGGTCC-3 ').
  • the vector pDLO1 2 was amplified with the aid of these oligonucleotides. After the T 4 DNA polymerase reaction with dCTP and dGTP, the ligation and subsequent transformation in £. coli, the positive clones could be identified by Xc / 77l digestion. The insertion of the interface was also checked by sequencing.
  • the bases marked with N are freely selectable, they do not contribute to the interface recognition. This enables the introduction of a sequence which is required for a Di / TriSec cloning via a certain often used cloning scheme. This has the advantage that only one Primer pair must be bought, which then allows cloning into different vectors.
  • the Xcl interface has the following sequence: Xcm ⁇
  • the trimming reaction (3'-5'-exonuclease activity) with the T4-DNA polymerase then takes place using dCTP as a stop nucleotide:
  • PCR fragments can then be cloned into the purified vectors, which have the sequence "TTC” at the 5 'end and "GAC” at the 3' end (in the example: G3BP cloning with the oligonucleotides 5'-G3BP-DLO ( 5'-TTC ATG GTG ATG GAG AAG CCT AGT-3 ') and 3'-G3BP (5'-GAC TTA CTG CCG TGG CGC AAG CCC CCT-3').
  • the PCR product has the following ends:
  • the vector and insert can be ligated.
  • the ligated DNA is transformed into E. coli.
  • the positive clones can be identified and checked using colony PCR and sequencing.
  • the DNA was then transformed into E. coli.
  • the positive clones could be identified by colony PCR and restriction digestion and verified by sequencing.
  • Example 4 Di / TriSec cloning of DNA oligonucleotides into a vector for in vivo expression of siRIMA
  • the developed Di / TriSec cloning is to be examined for its suitability for cloning DNA oligonucleotides in vectors which are suitable for the production of double-stranded RNA in transfected cells.
  • an inhibitor for the Lamin A / C gene should be generated.
  • oligonucleotides were mixed in different concentrations in a magnesium-containing buffer (see section 2.3.1) and heated to 95 ° C in a PCR machine.
  • the oligonucleotides were hybridized by slowly cooling from 95 ° C. over 3 hours to room temperature. The lid of the reaction vessel remained heated to 95 ° C.
  • the oligonucleotides can be dimerized in concentrations of 0.25-40 ⁇ M without this having a decisive influence on the ligation rate.
  • the pSUPER vector (Brummelkamp et al., 2002) was cut with the restriction endonucleases BglW and Hind ⁇ according to the manufacturer's instructions and filled in with dATP and dGTP using the method described above (see Section 2.1 .1).
  • the ligation was carried out as previously described (see section 2.3.2). The ratios of the free vector ends to the free insert ends of 1: 100 and 1: 500 were tested.
  • the ligation batches were transformed into E. coli using standard methods, and recombinant plasmids were propagated and purified.
  • the overall efficiency of the cloning of the double-stranded hLamin 1 IVE oligonucleotides was 84%, the vector to insert ratio having only a minor influence (1: 500, 80%, 1: 100, 86%).
  • the example shown here underlines the high efficiency of the cloning method according to the invention.
  • the minor influence of the Concentration of the oligonucleotides during the dimerization and the ratio of vector insert to the ligation rate demonstrate the robustness of the method in relation to the reaction conditions.
  • siRNA cloning technique shown here could represent an important step in creating a permanent "knockdown" cell clone library. All steps, starting with the dissolution of the oligonucleotides through the dimerization, the ligation, the transformation up to the PCR screen for positive clones and the isolation of plasmid DNA can be automated. As a result, silencer constructs for almost all human genes can be generated in a short time.
  • the advantage of the method described here over conventional methods is the high efficiency and robustness of the cloning.
  • Conventional strategies are based on the cloning of double-stranded oligonucleotides via palindromic restriction sites.
  • the oligonucleotides to be cloned preferably form concatamers. The result is a significantly reduced cloning efficiency.
  • the method described here excludes concatamer formation of the oligonucleotides and thereby increases the efficiency of the cloning.

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Abstract

La présente invention concerne des procédés et des ensembles de réactifs destinés à la manipulation d'ADN, notamment à la mutagenèse spécifique à la position ou au clonage.
EP02779456A 2001-10-02 2002-10-02 Procedes de manipulation d'adn Withdrawn EP1432797A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10148607A DE10148607A1 (de) 2001-10-02 2001-10-02 Methoden zur DNA-Manipulation
DE10148607 2001-10-02
PCT/EP2002/011086 WO2003031616A2 (fr) 2001-10-02 2002-10-02 Procedes de manipulation d'adn

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EP1432797A2 true EP1432797A2 (fr) 2004-06-30

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EP02779456A Withdrawn EP1432797A2 (fr) 2001-10-02 2002-10-02 Procedes de manipulation d'adn

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US (1) US20040248131A1 (fr)
EP (1) EP1432797A2 (fr)
AU (1) AU2002342790A1 (fr)
DE (1) DE10148607A1 (fr)
WO (1) WO2003031616A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009018449A1 (fr) 2007-07-31 2009-02-05 Verenium Corporation Assemblage combinatoire multi-sites sur mesure
ES2387167B2 (es) * 2011-02-21 2013-01-24 Universidade De Santiago De Compostela Nuevo método de clonación y mutagénesis in vitro mediante pcr inversa de clonación.

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580759A (en) * 1994-02-03 1996-12-03 Board Of Regents, The University Of Texas System Construction of recombinant DNA by exonuclease recession
US5700644A (en) * 1995-06-07 1997-12-23 Wisconsin Alumni Research Foundation Identification of differentially expressed genes
US5789166A (en) * 1995-12-08 1998-08-04 Stratagene Circular site-directed mutagenesis

Non-Patent Citations (1)

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Title
See references of WO03031616A3 *

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DE10148607A1 (de) 2003-04-10
WO2003031616A2 (fr) 2003-04-17
AU2002342790A1 (en) 2003-04-22
US20040248131A1 (en) 2004-12-09
WO2003031616A3 (fr) 2003-12-11

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