WO2000056876A1 - Mutagenese - Google Patents

Mutagenese Download PDF

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Publication number
WO2000056876A1
WO2000056876A1 PCT/GB2000/001075 GB0001075W WO0056876A1 WO 2000056876 A1 WO2000056876 A1 WO 2000056876A1 GB 0001075 W GB0001075 W GB 0001075W WO 0056876 A1 WO0056876 A1 WO 0056876A1
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selection
sequence
mutated
nucleic acid
mutation
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George Peter Lomonossoff
Li Liu
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Plant Bioscience Ltd
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Plant Bioscience Ltd
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids

Definitions

  • the present invention relates to methods and materials and kits for introducing mutations into target nucleotide sequences and recovering mutated material .
  • Site-directed mutagenesis is an important tool for the study of structure and functions of nucleic acids and proteins (see Ling & Robinson, 1997, for a recent review) .
  • Different strategies have been developed to perform the reaction and in particular to selectively recover mutated product material following the mutagenesis, which may represent as little as 1% of the reaction material .
  • ssDNA uracil-containing single-stranded DNA
  • dUMP uracil-containing single-stranded DNA
  • CJ236 uracil-containing single-stranded DNA
  • ssDNA is more time-consuming to obtain than double- stranded plasmid DNA (dsDNA)
  • dsDNA double- stranded plasmid DNA
  • selectable markers For those methods employing selectable markers, it is desirable that they do not require extra enzyme treatments, and that multiple rounds of mutagenesis can be performed without recloning target DNA.
  • One method which avoids recloning is that of Promega (1994) wherein two selectable markers are used. The method is performed by using two extra oligonucleotide sequences, one for repairing an ampicillin sensitive gene, and another one to inactivate the tetracycline resistant gene. Since this method needs two selection markers, the vector size is of necessity relatively large, and two selection steps are needed, which increases the time and reduces the efficiency of the process.
  • Another disadvantage of this method is that after the mutation reaction, it needs two round of transformations for selection of homologous clones .
  • the present inventors have devised a novel, rapid and efficient protocol which avoids the use of antibiotic mutation and repair but that can, if required, be used for multiple rounds of mutagenesis without recloning. Compared with the antibiotic selection method, it only needs one screenable, selection marker which permits the use of relatively small vectors (or allows the use of larger target sequences) .
  • homologous mutated clones can be obtained after only one round of transformation, using double-stranded plasmid DNA as templates, and bacterial strains commonly used in the art, wherein host cells carrying the desired mutation can be distinguished by colour alone.
  • the selection is carried out on the basis of mutation and repair of a selection nucleotide sequence encoding the -complement for ⁇ -galactosidase in conjunction with an appropriate chromogenic substrate.
  • the ⁇ -complement system in E. coli has per se been known for a considerable period of time (see e.g. Ullmann et al . , 1967; Gronenborn, 1976) .
  • the ⁇ -complement is an intra- allelic complement of a defective form of ⁇ -galactosidase encoded by a host, with the a ino-terminal fragment of ⁇ - galactosidase being encoded by a segment of DNA (LacZ) inserted in a suitable construct .
  • Bacteria which synthesize both fragments of the enzyme will produce blue colonies when plated on media containing the chromogenic substrate "X-gal', those which do not will be a 'white' colour.
  • the ⁇ -complement has previously been used in cloning (Messing et al . , 1977), including use as a control to check mutagenesis efficiency in many mutagenesis methods (for example, in Kunkel, 1985; Taylor et al . , 1985; Lewis an Thompson, 1990; Deng and Nickoloff, 1992). In these methods the aim is generally to check that the components being used in the method are functioning as they should.
  • the mutagenesis of the LacZ gene is not linked to that of the target sequence, and is generally carried out using a separate plasmid.
  • LacZ mutagenesis A different method employing LacZ mutagenesis is disclosed in WO 97/05484 (Beth Israel Hospital Association) .
  • a LacZ region based on the pUR288 plasmid, is integrated into the genome of an organism; the organism is subjected to mutagenesis techniques and then the LacZ region is recovered in circular form and mutation is investigated using a positive selection procedure in E. coli C (LacZ " ) .
  • the object of the method is to assess the efficiency of the mutagenesis method, with the LacZ itself being the 'target 1 .
  • the selection can be commenced on the basis of either repair of a deficient (e.g. frame-shifted) LacZ, or mutation of an active, read-through LacZ, and can proceed accordingly.
  • the cloning sites are positioned elsewhere in the vectors.
  • Two oligonucleotides controlling the LacZ expression and allowing the colonies exhibit blue or white colours are used as screenable primers . This property can be used for multiple rounds of mutagenesis by using the two screenable primers alternatively.
  • Figure 1, scheme (4) summarises the method.
  • a method of introducing a mutation into a target nucleotide sequence comprising the steps of: (i) providing a template nucleic acid construct comprising the target sequence and a selection nucleotide sequence encoding a selection polypeptide,
  • selecting a host carrying the product on the basis of expression of the selection sequence or the mutated selection sequence characterised in that the selection sequence is such as to allow simultaneous positive selection of a host expressing the selection sequence or a host expressing the mutated selection sequence
  • those hosts carrying a mutated selection sequence are selected since these will also comprise the mutated target sequence.
  • a method of introducing more than one mutation sequentially into a target sequence by using multiple rounds of mutagenesis comprising the steps of: (i) providing a product nucleic acid comprising a mutated target sequence and a mutated selection nucleotide sequence as described above,
  • the methods of the present invention differ from the use of antibiotic resistance markers in that the mutated selection sequence is not effectively a lethal mutation under the conditions used to select the non-mutated version.
  • the need for two separate markers each of which is positively selectable under different conditions is avoided.
  • site-directed mutagenesis generally uses circular ssDNA as wild-type template for one or more mismatch primers .
  • the primers are extended with a DNA polymerase to produce a double stranded DNA dsDNA product and the nick is ligated with DNA ligase.
  • the mutagenesis step in the methods above will initially yield a single stranded mutated "product' annealed to the template (a heterogenous product) .
  • this duplex may be used to generate a homogenous dsDNA construct corresponding to either double stranded "original 1 material or double stranded mutated "product' material.
  • the double stranded homogenous "product ' with the appropriate mutated selection sequence is preferably selected in appropriate host cells .
  • a host carrying the selection sequence and a host carrying the mutated selection sequence can be simultaneously positively selected, and distinguished, on the basis of colour.
  • the invention embraces other single (mutated) selection sequences which give non-lethal phenotypes to the hosts carrying them under the conditions used to select them.
  • either the selection sequence or the mutated selection sequence will encode an "active selection polypeptide" which is capable of altering the colour of the host in which it is expressed (or the vicinity thereof, in the case of extracellular enzyme activity) .
  • the selection sequence or the mutated selection not encoding the active selection polypeptide may, for instance, comprises a mutation which either prevents transcription of the sequence, or prevents translation of an active polypeptide from the transcribed sequence.
  • the mutation may optionally be a frame-shift mutation. However in each case the mutation is reversible with the appropriate primer.
  • the active selection polypeptide complements the activity of a host-encoded polypeptide, or is entirely new to the host.
  • the complemented activity of the host- encoded polypeptide is ⁇ -galactosidase activity.
  • the active selection polypeptide is the ⁇ - complement factor or an active variant thereof .
  • An ⁇ -complement "active variant” is one which is encoded by a derivative of the wild-type LacZ sequence which incorporates changes therein. Changes may be desirable for a number of reasons . For instance they may introduce or remove restriction endonuclease sites or alter codon usage. Alternatively changes to a sequence may produce a derivative by way of one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded ⁇ -complement polypeptide. Changes may be by way of conservative variation, i.e.
  • the active variant polypeptide is capable of complementing ⁇ - galactosidase activity in a suitable host as described above. It is reiterated that the starting point for the mutagenesis can be a vector encoding functional or non-functional selection polypeptide e.g.
  • selection nucleotide sequence encodes ⁇ -complement
  • this can either be functional or non-functional , the only proviso being that the "mutated selection sequence” encodes the opposite one.
  • the terms “mutated” and “reverted” are therefore relative and should be construed accordingly.
  • Example hosts include (but are not limited to) the E. coli strains JM109 and the commonly used DH5 ⁇ (or DH5 ⁇ F') which are discussed in the Examples below. It may be preferred to use a mis-match repair minus E. Coli host (e.g. XLblue mutS) in order to increase mutagenesis efficiency.
  • a mis-match repair minus E. Coli host e.g. XLblue mutS
  • dam ⁇ or dcm ⁇ strains may allow an increase in the ratio of desired colours in the whole colonies.
  • Other preferred hosts may be dut-, ung-bacteria .
  • the methods of the present invention will generally be preceded by the insertion of the target sequence into the template nucleic acid construct. This will generally be by means of a "polylinker" region in the construct i.e. one containing multiple restriction sites which is adapted to accept appropriately cleaved target sequences .
  • more than one target-mutagenesis primer can be used simultaneously to introduce more than one mutation into the target sequence in a single selection round. This may require the ligation of more than two polymerised nucleic acids .
  • the construct, or vector, used in the method may be any appropriate one suitable for site-directed-mutagenesis in the selected host .
  • Example vectors include plasmids based on the pBluescriptIISK(+) which are disclosed in the Examples below. As disclosed in the Examples, the mutagenesis may employ a dsDNA template.
  • Vectors adapted for use in the methods of the present invention form a further aspect of the invention.
  • the vectors will comprise an optionally mutated selection nucleotide sequence encoding a selection polypeptide, and a polylinker region.
  • the selection sequence is such as to allow simultaneous positive selection of a host expressing the selection sequence or a host expressing the mutated selection sequence as described above.
  • the vectors will be less than 5.8 Kbp in size, more preferably less than 5.0, 4.0, 3.0 Kbp, more preferably approximately 2.9 Kbp.
  • Example vectors include pM81B, pM81W, and pM83B. Vectors in which target sequence has been included (e.g. in the polylinker region) are also included, and naturally these could be much larger (e.g. 13 Kbp or more, as in pBinl9) .
  • a primer adapted for use in the methods of the present invention forms a further aspect of the invention.
  • These will be oligonucleotides , preferably of DNA although analogs may be used if preferred. Techniques for the synthesis of oligonucleotides can be found, amongst other places, in "Oligonucleotides and Analogues: A practical Approach” Ed. Eckstein, IRL Press, Oxford, (1992) .
  • Such primers will be either a mutagenesis primer capable of hybridising with the selection sequence such as to allow the introduction of a mutation therein (contains a mismatch) , or a repair primer capable of hybridising with the mutated selection sequence such as to allow reversion of the mutated selection sequence to the original selection sequence, in each case the selection sequences being as described above i.e. they give non-lethal phenotypes to the hosts carrying them under the conditions used to select them.
  • Pairs of such primers are particularly preferred.
  • Example primers include W2B and B2W:
  • W2B (white to blue, 5' CCAGTGAGCGCGATCGCGTAATCATGG 3').
  • kits for performing the methods of the present invention may include the primers and vectors discussed above.
  • kit for introducing a mutation into a target nucleotide sequence comprising:
  • a template nucleic acid construct comprising an optionally mutated selection nucleotide sequence encoding a selection polypeptide, and a polylinker region,
  • a mutagenesis primer capable of hybridising with the selection sequence such as to allow the introduction of a mutation therein
  • a repair primer capable of hybridising with the mutated selection sequence such as to allow reversion of the mutated selection sequence to the original selection sequence, characterised in that the selection sequence is such as to allow simultaneous positive selection of a host expressing the selection sequence or a host expressing the mutated selection sequence
  • the primers and vectors may be those described above.
  • the kit may comprise further materials for ligating target sequences, for performing the polymerisation, or for detecting the selection polypeptide. Specific details of kit materials suitable for the polymerisation are found in commercially available kits e.g. as described in current Catalogues or Websites of Amersham, Pharmacia, Strategene, Clonetech, Bio-Rad, and Promega . See also WO 97/20950 or references disclosed therein.
  • Ligase T4 DNA ligase
  • dNTP mix (1.25 mM dATP, dCTP, dGTP, dTTP, 2.5 mM ATP and 35 mM MgCl 2 )
  • Host cells e.g in freeze dried form, or pre-made competent cells in glycerol
  • Chromagenic substrates e.g. X-Gal 50 mg/ml ; IPTG
  • Buffers for annealing; synthesising; enzyme dilution
  • the invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.
  • Figure 1 shows a schematic diagram of a method in accordance with the present invention - Scheme (4) .
  • the other schemes represent the prior art.
  • Scheme (1) is based on restriction site elimination
  • (2) is a replication defect protocol
  • (3) is an antibiotic repair protocol with two antibiotic markers.
  • Figure 2 schematically shows the result of a typical "blue to white” protocol resulting in double stranded heterogenous and homogenous products.
  • the " shows the mutations introduced.
  • A is the target;
  • b is the sceenable selection marker.
  • the mutant product gives white colonies on X-gal and IPTG.
  • the heterozygous blue double stranded product shown in the Figure can be avoided.
  • Figure 3 shows a scheme of various vector construction and mutagenesis reactions according to the present invention, as described in the Examples below.
  • Functional LacZ is shown as a block.
  • Deficient LacZ is shown in outline. Also shown are cloning sites and insertions. Mutated sites (other than in Lac Z) are shown by a dark circle.
  • Example 1 Mutation efficiency by selection based on white colonies to blue colonies
  • MutS strains were used for transformation, and the cells were incubated for two to three hours in liquid culture and then transferred to petri dishes. After overnight, single colonies containing the desired screenable marker were picked up. Up to 60% of colonies were homologous and contained the targeted mutations.
  • Example 2 Mutation efficiency by selection based on blue colonies to white colonies
  • pM81B was mutagenized by using the B2W and a target primer. Two mutagenesis reactions were performed and 50 to 63 percent of colonies contained the desired mutations (Table 1, Reaction C and D) .
  • nucleotide (s) 1 nt for nucleotide (s) .
  • the simultaneous mutation rate is 60 percent.
  • Plasmid DNA of pM81W2.4K and pM83B0.7K was mutagenised by using target primers and W2B or B2W.
  • the mutagenesis rate is 70 to 100 percent analysed by restriction digestion for pM81B2.4K and sequencing for pM83W0.7K (Table 1, Reaction E and F) .
  • mutagenesis oligonucleotides were used to mutagenize pM83B0.7K and pM81W2.3K, and four target primers were used for pM81W2.4K.
  • the simultaneous mutation rate is from 60 to 75 percent for two-site samples, and 38 percent For the four- site sample (Table 1, Reaction G, H, and I) .
  • T4 and T7 DNA polymerases were tested in order to find the best combination. Mutated DNA was transformed and directly transferred to Petri dishes to check the blue/white ratio. With template DNA from different E. coli hosts, the enzyme effects were not the same. In general, results showed that when T4 DNA polymerase was used, mutated colonies (blue/white ratio) were usually less than 0.1%. When T7 DNA polymerase was used, the enzyme appeared to reduce the total number of colonies at higher concentrations (Table 2) .
  • Example 8 effect of ratio of screenable and target oligonucleotides
  • Oligonucleotide ratio and amount may affect the efficiency of targeted mutagenesis.
  • the amount of screenable and target oligonucleotides and its effect on targeted mutation efficiency were investigated. The results showed that both oligonucleotides at 4 pmol produced best targeted mutations (Table 3) .
  • Plasmid DNA containing insertions from 2 kbp to 6.0 kbp was used to test the target mutation rate over marker mutation.
  • the mutagenesis rate was 25 to 71 percent analysed by restriction digestion analysis (Table 4) .
  • Example 10 further example of multiple simultaneous mutations
  • two or four target mutagenesis oligonucleotides were used in addition to a screenable oligonucleotide.
  • the simultaneous mutation rate was 58 percent for the two-site mutation sample, and 16 percent for the four- site sample (Table 5) .
  • Example 11 effect of incubation time on the segregation of cells containing heterologous DNA
  • the invention provides, inter alia, an efficient and rapid site-directed mutagenesis method using ⁇ -complement for colour selection as a screenable marker.
  • This new method has several advantages over current approaches:
  • Normal dsDNA may be used as templates compared to Kunkel ' s method which uses only uracil-containing ssDNA (Kunkel 1985; Kunkel et al . , 1991) .
  • Enzyme digestion is not necessary after the reaction, which is a vital step for successful unique site elimination method (Deng & Nickoloff , 1992) .
  • DNA from individual colonies containing a marker gene may be used for the second transformation, thus eliminating the possibility that plasmids selected from the second transformation are multiplied from same colonies.
  • the procedure can employ high fidelity T4, or more preferably T7, DNA polymerase which reduce the unwanted secondary mutations that are often encountered in PCR-based mutagenesis. Additionally, T7 polymerase gives fast synthesis and high processivity (Tabor et al , 1987) which is useful when using large inserts. T7 was also found to increase the ratio of mutated screenable colonies, by reducing the activity of unmutated DNA.
  • Restriction enzymes were purchased from Boehringer, GIBCO-BRL or New England BioLabs.
  • T4 DNA polymerase and polynucleotide kinase were from GIBCO-BRL or Pharmacia.
  • T4 DNA ligase was from Boeringer .
  • T7 DNA polymerase was from New England.
  • E. coli strain DH5 ⁇ and DH5 ⁇ IQ was from GIBCO-BRL, JM109 from Promega, XLblue mutS from Stratagene. X-gal and IPTG from Pharmacia. DNA purification kits and columns were from Promega .
  • the pMO was constructed by deleting the .BssHII fragment in pBluescriptIISK(+) .
  • the mutagenesis method was tested by using W2B (white to blue, 5' CCAGTGAGCGCGATCGCGTAATCATGG 3') and V4514, an oligonucleotide of 35 nucleotides, to mutate dsDNA or ssDNA of pMO to pMOB, in which a additional -Seal was created at the LacZ gene by W2B and a Ascl site downstream of the LacZ gene by V4514.
  • ssDNA of pMO was mutated by using B2W (blue to white, 5' CCAGTGAGCGCGCTTGGCGTAATCATGG 3') and V4514, in which restriction sites -Bgr-lII was produced at the LacZ gene by B2W, and a Ascl site downstream of the LacZ gene by V4514.
  • the LacZ gene was reading through in pMOB and was disrupted in pMOW. When these vectors were transformed into LacZ negative bacteria strains, colonies will be show blue or white, respectively.
  • a 2.4 Kbp fragment consisting of CaMV 35S promoter and a cDNA fragment of cowpea mosaic virus RNA1, and a 2.3 Kbp fragment containing a NPTII gene were cloned into pM81W to produce pM81W2.4K and pM81W2.3K, respectively.
  • a 0.7 Kbp fragment containing a cDNA fragment of red clover mosaic virus was cloned into pM83B (a derivative of pM81B) to produce pM83B0.7K.
  • the dsDNA of constructs were applied as templates .
  • One or more mutagenic primers together with W2B or B2W were used to perform mutagenesis.
  • the oligonucleotides were synthesized on the ExpediteTM Nucleic Acid Synthesis System (Millipore) .
  • the DNA was treated with 35% NH 4 0H, precipitated with 70% ethanol and dissolved in distilled water. The concentration was determined by spectrophotometer .
  • Phosphorylation were performed at 37°C for 30 minutes and stopped by incubating at 70°C for 10 minutes in a 25 ⁇ l of volume containing 4 pmol/ ⁇ l primer, 50 mM This-HCl, pH7.6 , 10 mM MgCl 2 , 5 mM dithiothreitol , 0.1 mM spermidine HC1 , 0.1 mM EDTA, pH8.0 , 0.8mM ATP, 10U T4 DNA polynucleotide kinase .
  • the dsDNA was purified using the alkaline method (Sambrook et al . , 1989) and further treated using a DNA purification kit. Purified DNA was adjusted to 0.05 pmol/ ⁇ l. Mini-prep DNA (Sambrook et al . , 1989) was sometimes used to test the adaption of the system.
  • mutagenesis was performed as follows. 0.05 pmol of template DNA was mixed with 4 pmol of screenable W2B or B2W primer, 4 pmol of target primer (s) in a 20 ⁇ l of volume containing 20 mM Tris-HCl, pH7.5 , 10 mM MgCl , 50 mM NaCl . The mixture was incubated in a 100°C water bath for 3 minutes, and immediately placed on ice for another 3 minutes.
  • mutagenesis was performed as follows, if not indicated otherwise. An aliquot (0.05 pmol) of column- cleaned DNA (1 ⁇ l) or 15 ⁇ l of mini-prep DNA was placed in a 0.5 ml micro-centrifuge tube, to which 4 ⁇ l of 2N NaOH, and H 2 0 to a total volume of 20 ⁇ l were added. The mixture was incubated at room temperature for 10 minutes to denature the DNA, and 10 ⁇ l of 3M sodium acetate, pH5.2 and 100 ⁇ l of ethanol were added. The sample was then placed on dry ice for
  • the sample was added with 300 ⁇ l of LB, incubated at 37°C for 10 to 20 minutes and transfer to Petri dish containing LB, carbenicillin, X-gal and IPTG.
  • the screenable mutating colonies were recorded after 20 hours according to the colour, and targeted mutations were further tested by enzyme digestion of purified DNA.
  • the sample was treated as follows. After 90 seconds on ice, the cells were transferred to 10 ml of LB and the mixture incubated at 37°C for one hour. Antibiotic (carbenicillin) was the added and the cells incubated for a further tow hours under the same conditions before transfer to a Petri dish. Blue or white colonies were selected according to screenable oligonucleotide for confirmation of target mutations.

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Abstract

L'invention concerne des techniques permettant d'introduire des mutations dans une séquence nucléotidique cible. Cette technique facilite l'introduction séquentielle de plus d'une mutation dans une séquence cible à l'aide de plusieurs séries de mutagenèses, ce qui permet de sélectionner positivement les séquences mutées appropriées après chaque étape. On peut utiliser simultanément plus d'une amorce cible pour introduire plus d'une mutation dans la séquence cible. De préférence, les hôtes portant la séquence de sélection et la séquence de sélection mutée peuvent être simultanément sélectionnés et distingués de manière positive, en fonction de la couleur provenant par exemple de mutations dans le facteur de complément alpha, ou dans un variant actif de celui-ci codé par exemple par lacZ.
PCT/GB2000/001075 1999-03-23 2000-03-22 Mutagenese Ceased WO2000056876A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP1626978A4 (fr) * 2003-02-06 2007-05-02 Rensselaer Polytech Inst Protocoles bases sur la polymerase pour l'introduction de deletions et d'insertions

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WO1997020950A1 (fr) * 1995-12-08 1997-06-12 Stratagene Mutagenese amelioree, dirigee sur un site d'un adn circulaire
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EP0319759A2 (fr) * 1987-12-11 1989-06-14 Abbott Laboratories Méthode de mutagénèse par réparation de rupture de chaîne dirigée par une oligonucléotide
WO1991006643A1 (fr) * 1989-10-27 1991-05-16 Majesty (Her) In Right Of Canada As Represented By The National Research Council Of Canada Procede de mutagenese specifique au site
US5955363A (en) * 1990-01-03 1999-09-21 Promega Corporation Vector for in vitro mutagenesis and use thereof
WO1993013216A1 (fr) * 1991-12-24 1993-07-08 The President And Fellows Of Harvard College Mutagenese d'adn dirigee
WO1997020950A1 (fr) * 1995-12-08 1997-06-12 Stratagene Mutagenese amelioree, dirigee sur un site d'un adn circulaire
WO1998002537A1 (fr) * 1996-07-17 1998-01-22 Promega Corporation Mutagenese dirigee et selection de mutants a l'aide de marqueurs antibio-resistants codant des produits geniques presentant une specificite de substrat modifiee

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Publication number Priority date Publication date Assignee Title
EP1626978A4 (fr) * 2003-02-06 2007-05-02 Rensselaer Polytech Inst Protocoles bases sur la polymerase pour l'introduction de deletions et d'insertions

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