CN1656221A - Method for creating polynucleotide molecules - Google Patents
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- CN1656221A CN1656221A CNA038119277A CN03811927A CN1656221A CN 1656221 A CN1656221 A CN 1656221A CN A038119277 A CNA038119277 A CN A038119277A CN 03811927 A CN03811927 A CN 03811927A CN 1656221 A CN1656221 A CN 1656221A
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Classifications
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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
-
- 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
-
- 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
- C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR
Abstract
The invention relates to methods for creating polynucleotide molecules with modified characteristics, said methods running through a cycle comprising the following steps:(a) provision of a double-stranded polynucleotide molecule or a population of double-stranded polynucleotide molecules, the individual polynucleotides of said population having at least one homologous sequence part and at least one heterologous sequence part,(b) creation of single-strand breaks in the double-stranded polynucleotide molecules, (c) nucleolytic degradation in the 5'} 3' direction starting from the single-strand breaks with simultaneous new synthesis in the 5' } 3' direction and shifting of the single-strand breaks in the direction of the 3' end, (d) production of single-stranded polynucleotide molecules, (e) production of partially double-stranded polynucleotide molecules of the single-stranded polynucleotide molecules provided in step (d), (f) template-directed nucleic acid synthesis starting from the partially double-stranded polynucleotide molecules produced in step (e), whereby steps (b) and (c) can be carried out in succession or simultaneously.
Description
The present invention relates to produce method with the polynucleotide molecule that changes characteristic.
Understand as us, biomolecules, especially biopolymer as polynucleotide, polypeptide, polysaccharide etc., not only form the basis of organism, and obtain day by day using in whole industrial application.The searching of new functionalized biomolecules, separation and preparation and their industrial application are the themes of modern biotechnology.(reference: the screening crude substance), occurred the method for simulating nature evolution principle in the laboratory recently the biomolecules unknown, that have desired characteristic before chancing on occurring in nature.
Except the method that produces point mutation (in the mode of base exchange, disappearance and insertion), to the structural domain of polymer inside, subunit or the genetic mutation of gene cluster inside or the gene of genome inside of heteromultimers, the reorganization of sequence part comes down to be used for the extremely successful strategy of combined spot sudden change.Homologous recombination, promptly from the combination of the corresponding sequences part of different variants and keep direction and reading frame constant, be particularly important.
Experimentally, reorganization may be implemented in a variety of ways: the first, and external by using independent enzyme function or the mixture of determining or the enzyme treatment step of sequence; The second, recombinate and/or repair procedure by the use cell in the body.
In the past, mainly use the method for PCR-based at industrial in vitro method.Here at first should be mentioned that DNA reorganization (DNA shuffling), (WO 95/22625 to be also referred to as secual PCR; Stemmer, Nature 370 (1994), and 389).In the method, sequence eclipsed random gene fragment obtains producing, and rebuilds the product that obtains original length by no primer PCR subsequently.Therefore, in each PCR circulation, by mutual initiation, the fragment of different sources randomly and to be combined into the product molecule with the seedbed be possible.By the adjustment sheet segment length, the frequency that DNA reorganization limits recombination event in principle is possible.Yet this method is complicated experimentally, because at first must set up the reaction conditions that produces nucleotide fragments.Shao etc. (Nucl.Acids Res.26 (1998), 681) have reported another external method for preparing recombinant DNA.This method is used the primer with stochastic sequence, and this primer can make polyreaction begin in the site at random of polynucleotide inside.Therefore, similar with DNA reorganization, produce short polynucleotide passage, these fragments can be recombinated by mutual initiation.Use this method may control recombination frequency hardly.In addition, non-special primer causes high relatively inherited error rate, and this can become the problem of responsive sequence part and/or long gene.Alternative as these methods in pcr amplification, is to promote the chain exchange, and (WO 98/42728 to use improvement PCR method to carry out the method for staggered extension; Zhao etc., Nat.Biotechnol. (1998), 258).By use unwind and annealing stage between the polymerization temperature in very short stage, not exclusively product can be hybridized with new template, and can further extend.Recombination frequency can be adjusted by presetting polymerization time and cycle number.Here technical restriction is the very short stage that need accurately be set under the specified temp.Alternative as these PCR-based methods, a kind of like this method has been described, this method can produce heteroduplex from the colony of polynucleotide with sudden change, these heteroduplexs then by with behind their transfered cells through repairing at random in the body, or external after they and cell extract hatched repaired at random, rely on the relative frequency of variant in the initial colony, and cause producing a certain proportion of recombinant molecule variant (WO 99/29902).This method is feature to use the cytothesis system, but this system's specific recognition is not matched base and repair any chain in the two strands at random.This method of one side is subject to the controllability that lacks in the limited efficient of polynucleotide transfered cell and the repair process.In addition, a conclusive shortcoming is to repair step, the fact that only has two starting molecules to recombinate mutually at each.
An object of the present invention is to provide the method that preparation has the polynucleotide that change characteristic, this method can be avoided the above-mentioned shortcoming of known method, and can allow the effective reorganization between the polynucleotide molecule genotype, thus mutagenic phenotype.
We find that this purpose can realize by the embodiment described in the claim is provided.
Therefore, the present invention relates to produce the method with the polynucleotide molecule that changes characteristic, this method comprises at least one circulation that comprises the steps:
(a) provide a kind of double-stranded polynucleotide molecule or the colony of double-stranded polynucleotide molecule is provided, independent polynucleotide have at least one homologous sequence part and at least one heterologous sequence part in the wherein said colony,
(b) in described double-stranded polynucleotide molecule, produce the strand breach,
(c) begin to carry out 5 '-3 ' nuclease degradation from described strand breach, 3 ' end at described strand breach carries out 5 '-3 ' de novo synthesis and mobile simultaneously,
(d) preparation strand polynucleotide molecule,
(e) the partially double stranded polynucleotide molecule of the strand polynucleotide molecule that provides of preparation process (d),
(f) initial to carry out template guided nucleic acid synthetic from the partially double stranded polynucleotide molecule of step (e) preparation,
Can be successively or implementation step (b) and (c) simultaneously.
Therefore, method of the present invention can be by being distinguished in conjunction with its advantage, and this former described arbitrary method all can not realize.The aforesaid method additional advantage is its low possibility of testing complicacy and only needing a spot of time and automatization is also arranged.
Method of the present invention can be distinguished by such fact, and this fact is to exist nuclease degradation and nucleic acid synthetic simultaneously, i.e. and " nick translation ", but with the excessive fragmentation of avoiding nucleic acid and the degraded of recombinant nucleic acid.In principle, all DNA amount of use can be used for the vitro recombination of described nucleic acid subsequently.
Compare with the method for in the past described external nucleic acid reorganization, this makes that increasing recombination efficiency becomes possibility.
Embodiment preferred comprises the circulation of carrying out comprising more than one above-mentioned steps (a)-(d), promptly at least 2, preferred at least 5, particularly preferred at least 10 and at least 20 circulations very particularly preferably.
Therefore, the cycle applications of the inventive method makes that having the polynucleotide that make up sequence area for several times recently from the initial distribution preparation of related polynucleotides sequence becomes possibility.Described cycle applications especially allows the combination mutually of multiple different heterologous sequences part.In addition, can accurately control the recombination frequency of each polynucleotide chain by cycle number.Cycle applications also makes to control by this way from a Mean Time Between Replacement that is circulated between the next round-robin recombination event becomes possibility.
In a further preferred embodiment, select step after one of the inventive method, several or all circulations, to carry out.Described selection step may with genotype or phenotype or all relevant with the genotype and the phenotype of polynucleotide.
In this article, the genotype of polynucleotide is sequences of described polynucleotide different monomers.Phenotype is the function of transcribing of polynucleotide molecule and polynucleotide encoding or translation product and the summation of characteristic.
For example, select step to realize, select by physical sepn, or select (Koltermann and Kettling, Biophys.Chem.66 (1999), 159 by screening by selection with amplification coupling (nature); Kettling etc., Current Topics in Microbiol.and Immunol.243 (1999), 173; Koltermann, Dissertation, TU Berlin (1998), Zhao etc., Manual of Ind.Microbiol.and Biotechnol.49 chapter, 597-604 page or leaf, ASM Press, Washington, DC, 1999; Reetz, Angew.Chem.113 (2001) 113,292-320).The step of the inventive method (a) provides the colony of a kind of double-stranded polynucleotide molecule or double-stranded polynucleotide.
The colony of the double-stranded polynucleotide molecule that provides according to the step (a) of the inventive method can be to comprise containing of at least two kinds of types at least a homologous sequence part and the arbitrary double-stranded polynucleotide molecule colony of at least a heterologous sequence polynucleotide molecule partly.In this article, term " strand polynucleotide molecule colony " refers to a certain amount of such polynucleotide molecule, and the molecular interaction that these molecules take place with specificity base pairing form is prevented from or does not exist.The term here " polynucleotide " (nucleic acid, oligonucleotide) comprises DNA and RNA, and polynucleotide are the strand of linear, directed (5 '-3 ') or the heteromultimers of double chain form.In two strands, by the interaction of specificity base pairing, two strands are bonded to each other.In principle, polynucleotide can be to have modified monomeric DNA or RNA.Generally speaking, present method also can be used for the same artificial polymer that makes up and is used for the DNA-RNA heteroduplex.
Term " homology part " refers to identical or complementary part in two or more polynucleotide molecules, promptly comprises identical information in corresponding site.Term " allos part " refers to inequality or complementary part not in two or more polynucleotide molecules, promptly comprises different information in corresponding site.Here the information of polynucleotide molecule (genotype) refers to the sequence of different monomers in the polynucleotide molecule.The length in heterologous sequence zone has a Nucleotide at least, but in fact can be longer.The heterologous sequence zone especially can be two Nucleotide or three Nucleotide, codon for example, and, particularly preferably be the Nucleotide more than 10 at the Nucleotide more than 5 preferably on the length.In principle, the length in allos zone does not have the upper limit.Yet preferably, the allos zone should be no longer than 10000 Nucleotide, and is particularly preferred no longer than 5000 Nucleotide, especially no longer than 2000, very particularly preferably no longer than 1000 Nucleotide.This relatively-long sequences part can be, for example, and the gene of the hypervariable region of encoding antibody sequence, proteinic structural domain, gene cluster or genomic zone.The allos zone preferably wherein polynucleotide molecule at each mutually different sequence area in base place.Yet the allos zone also can be disappearance, repetition, insertion, upset or the interpolation that has occurred in polynucleotide molecule or taken place.
Double-stranded polynucleotide molecule according to the step (a) of the inventive method provides according to the present invention, has at least one homology and at least one heterologous sequence zone.Yet preferably, they have a plurality of homologies and allos part.In principle, the quantity of homologous region and allos part does not have the upper limit.
Allos part in the above-mentioned double-stranded polynucleotide molecule partly is interrupted by homology under each situation.In this article, preferably at least 5 of the length of homology part, preferably at least 10, and particularly preferred at least 20 Nucleotide.Yet similar as the allos part, the homology part in fact can be longer, and their length does not have the upper limit in principle.Preferably, they are particularly preferred no longer than 10000 Nucleotide preferably no longer than 20000 Nucleotide no longer than 50000 Nucleotide, and very particularly preferably no longer than 1000 Nucleotide.
By the known method of skilled work personnel, double-stranded polynucleotide molecule can provide according to the step (a) of the inventive method.These comprise, for example physics, chemistry, biological chemistry and biological method.These comprise synthetic and preparation method, as, for example chemosynthesis of oligonucleotide, by polymerase chain reaction (PCR) nucleic acid, preparation plasmid, clay, phage, BACs (bacterial artificial chromosome), YACs (yeast artificial chromosome) or chromosomal DNA.
In the inventive method particularly preferred embodiment, the colony with double-stranded polynucleotide of homology and allos part provides by the related polynucleotides sequence that use comes from the quasispecies mutant.The term here " is correlated with " and is related to the polynucleotide that have homology and allos district.Quasispecies refers to the dynamic population of inter-related molecular variants (mutant), duplicates by fallibility and can produce molecular variants.Displayablely be, according to the quasispecies principle, the object of selection is not wild-type (barycenter of quasispecies), but whole distribution.Along with the selection condition that changes, according to their fitness value, this mutant distributes and has comprised useful variant, does not therefore need at first the random mutation by subsequently to produce.Under the situation of selecting parameter to change in succession, the generation of evolution is similar to described quasispecies along specific value directional drift stealthily.WO 92/18645 has described the preparation of quasispecies and the application of evolution biotechnology principle.
If the fallibility based on molecular variants duplicates, can produce quasispecies.When using polynucleotide, to duplicate and can preferably under the help of replicative enzyme, carry out, described replicative enzyme is the polynucleotide molecule synthetic polysaccharase of template control.The introducing of mistake, be the variation of molecular information, can be only by intrinsic fallibility copy procedure or other process by special increase polysaccharase mistake (for example specificity is added monomer lopsidedly, added base analogue, fallibility PCR, polysaccharase) with high error rate, by polynucleotide after synthetic chemically modified, by using monomer mixture and/or nucleotide analog to carry out the synthetic fully of polynucleotide to small part and the combination by aforesaid method realizes.The preferred distribution of using the quasispecies mutant, wherein the phenotypic characteristic of the target molecule function of each mutant of above-mentioned quasispecies is compared with wild-type and is improved.Term " phenotype of polynucleotide molecule " refers to the function of transcribing of polynucleotide molecule and polynucleotide encoding or translation product and the summation of characteristic.
In addition, can use the sequence of different sources, especially derive from the polynucleotide sequence of the gene family of different plant species, the polynucleotide sequence that duplicates with extra high error rate is (for example by virus in vivo, by having the bacterium of mutator gene, by the bacterium under the uviolizing), or at the external polynucleotide sequence that duplicates with extra high error rate (for example by Q β replicative enzyme reaction, the method of fallibility PCR), introduce sudden change or have the polynucleotide sequence of the mode chemosynthesis in homology and allos district by chemical reagent after synthetic with them, or the polynucleotide sequence of the combination results by aforesaid method.In principle, the employed polynucleotide of the inventive method can be arbitrary polynucleotide, especially DNA or RNA molecule.
The needed strand breach of the step of the inventive method (b) can produce by arbitrary method in principle, and these methods can cause in the polynucleotide chain of double-stranded polynucleotide molecule the phosphodiester bond between 2 Nucleotide to disconnect.Aforesaid method can be method physics or chemistry (for example esterolysis of supersound process, part).
Enzyme method is particularly useful for step (b).
The example that is applicable to the enzyme of this situation is a nuclease.
In the inventive method embodiment preferred, the strand breach is introduced by sequence-specific nickase.
The example of above-mentioned nickase is the V.BchI that comes from erosion chitin genus bacillus (Bacillus chitinosporus), the N.BstNBI that comes from bacstearothermophilus (Bacillus stearothermophilus), the N.BstSEI that comes from bacstearothermophilus, the N.CviPII that comes from green alga (Chlorella) NC64A strain, the N.CviQXI that comes from green alga NC64A strain, the V.EcoDem that comes from intestinal bacteria (E.Eoli), the V.HpaII that comes from haemophilus parainfluenzae (Haemophilusparainfluenzae), come from the V.XorII that gas is given birth to the V.Neal of bacterium colony Nocardia bacteria (Nocardiaaerocolonigenes) and come from rice Xanthomonas (Xanthomonas oryzae).
In a further preferred embodiment, can in above-mentioned double-stranded polynucleotide molecule, introduce the strand breach by non-sequence-specific nickase.In this article, can use, for example with Mg
2+As the calf pancreas DNase I of cofactor (Kunitz, J.Genetic Physiology 33 (1950), 349; Kunitz, J.Genetic Physiology 33 (1950), and 363 and Melgac and Goldthwaite, J.Biolog.Chem.243 (1968), 4409).
According to the reaction conditions in the enzyme selection step of using (c).
In preferred embodiments, step (c) is carried out can causing increasing under the condition of de novo synthesis error rate.
Can select the error rate of above-mentioned de novo synthesis according to the target variant that desire produces.Typical error rate is 0.1 * 10
-3-10 * 10
-3, i.e. the mistake of 0.01-1% (in the DNA of 10000 bases part, having changed 1-10 base).
It is useful especially carrying out step (c), and this step has error rate 1 * 10
-3-5 * 10
-3, promptly the mistake of 0.1-0.5% promptly in the DNA of 1000 bases part, has changed 1-5 base.
The error rate of dna polymerase i is 9 * 10
-6(Kunkel etc. (1984) J.Biol.Chem.259:1539-1545).Therefore, when using dna polymerase i, the increase of error rate is exactly that error rate surpasses 9 * 10
-6
In principle, the error rate of de novo synthesis can increase, for example by using the archaeal dna polymerase that suddenlys change or passing through to select appropriate reaction conditions in the step (c).
In preferred embodiments, the error rate of de novo synthesis can have the polysaccharase that reduces or do not have proofreading activity by use and obtains increasing.
In the preferred embodiment of the inventive method, the error rate of de novo synthesis can obtain increasing as initial substance by the Nucleotide with different concns.In this article, the concentration of every kind or several nucleotides can obtain changing with respect to other Nucleotide.Compare preferably substoichiometric a kind of Nucleotide, especially dATP with other Nucleotide.The example of proper concn is that dGTP, dCTP and dTTP are 200 μ M, and ATP is 20-50 μ M.
Compare preferably substoichiometric two kinds of Nucleotide, especially dATP and dGTP with other Nucleotide.The example of proper concn is that dCTP and dTTP are 200 μ M, and dATP and dGTP are 40 μ M.
In the further embodiment of the inventive method, the error rate of de novo synthesis can obtain increasing by adding nucleotide analog.The nucleotide analog of mentioning is dITP, 7-denitrogenation deoxyguanosine triphosphate and dezyribonucleoside α-thio triphosphates.The particularly preferred dITP that is to use.
In the further embodiment of the inventive method, the error rate of de novo synthesis can obtain increasing by changing salt concn.The example that is applicable to this situation is with Mg
2+Ionic concn increases to more than the 1.5mM.What also be suitable for is to add Mn
2+Ion, for example concentration is 0.1-1mM, especially 0.2-0.5mM.
In the further embodiment of the inventive method, the error rate of de novo synthesis can obtain increasing by adding additive.The additive that is fit to is any material that increases error rate, and the example of mentioning is dimethyl sulfoxide (DMSO), polyoxyethylene glycol and glycerine.Particularly preferably be by following concentration and add above-mentioned additive: DMSO 2-10%, PEG 5-15%, glycerine＞0-30%, preferably 5-20%.
In further embodiment, the error rate of de novo synthesis can obtain increasing by changing temperature of reaction, especially elevated temperature.
All methods of mentioning for the increase error rate also can make up enforcement, for example Mn mutually
2+Nucleotide was excessive when ionic concn increased.
In preferred embodiments, step (b) and (c) carried out simultaneously.
According to the step (d) of the inventive method, the preparation of strand polynucleotide molecule can be accomplished by using the known method of skilled work personnel.These methods comprise, for example physics, method chemistry, biochemical and biological.Here the example of listing is two strands (Newton, PCR, the Spektrum AkademischerVerlag (1994) that unties polynucleotide more than the annealing temperature by temperature is heated to; Lazurkin, Biopolymers 9 (1970), 1253-1306), by adding denaturing agent (for example urea or stain remover) with the double-stranded sex change of polynucleotide, add the enzyme that double-stranded polynucleotide can be changed into the strand polynucleotide, for example change double-stranded DNA into single stranded DNA by the circumscribed degraded of nuclease.
The preparation of the partially double stranded polynucleotide molecule of the strand polynucleotide molecule that step (d) provides, step (e) according to the inventive method, can use the known method of skilled work personnel and accomplished, and can be preferably accomplished by the hybridization of complementary strand polynucleotide molecule homology part.
The hybridization that produces double-stranded polynucleotide uses the known method of skilled work personnel to carry out, especially, for example can be by realizing that in conjunction with strand and setting reaction conditions this can pass through, as, for example reduce temperature and/or salt concn and promote the annealing of complementary polynucleotide.
From the partially double stranded polynucleotide molecule of step (e) preparation, template guided nucleic acid synthesizes realization in the step (f) in the inventive method.
The term here " template guided nucleic acid is synthetic " refers on the basis of corresponding templates chain information the synthetic polyribonucleotides by the extension of existing strand.
Skilled staff is ripe in carrying out this template guided polyreaction, for example described in the Sambrook (Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)).
But have the polynucleotide polymerization activity of template control and arbitrary enzyme of synthetic polyribonucleotides chain and can be used for polymeric enzyme reaction.In a large number obtained separating and describing from multiple organism and polysaccharase with difference in functionality.Between the RNA polymerase (replicative enzyme) that archaeal dna polymerase that DNA relies on, archaeal dna polymerase (reversed transcriptive enzyme) that RNA relies on, RNA polymerase that DNA relies on and RNA rely on, there is difference about template and synthetic polynucleotide type.About temperature stability, between the polysaccharase of non-thermally-stabilised (37 ℃) and thermally-stabilised (75-95 ℃), there is difference.In addition, polysaccharase also is different with regard to 5 '-3 ' and 3 '-5 ' exonuclease activity that exists.Most important polysaccharase is the archaeal dna polymerase that DNA relies on.
Especially, can use and have optimum temperuture and be approximately 37 ℃ archaeal dna polymerase.These polysaccharases comprise, the T4 archaeal dna polymerase of the T7 archaeal dna polymerase of e. coli dna polymerase I, phage t7 and phage T4 for example, each all has a large amount of manufacturerss for example USB, RocheMolecular Biochemicals, Stratagene, NEB or Quantum Biotechnologies sells commercial.E. coli dna polymerase I (holoenzyme) has 5 '-3 ' polymerase activity, 3 '-5 ' gauged exonuclease activity and 5 '-3 ' exonuclease activity.(Rigby etc. (J.Mol.Biol.113 (1977)), 237-251), this enzyme is used for external dna marker by the nick translation method.Opposite with holoenzyme, the Klenow fragment of e. coli dna polymerase I, the same as the T7 archaeal dna polymerase with the T4 archaeal dna polymerase, there is not 5 ' exonuclease activity.Therefore, these enzymes be used for " filling and leading up reaction " or synthetic long-chain (Young etc. (and Biochemistry 31 (1992), 8675-8690), Lehman (Methods Enzymol.29 (1974), 46-53)).At last, 3 '-5 '-exo (-) variant of the Klenow fragment of e. coli dna polymerase I also lacks 3 ' exonuclease activity.This enzyme is usually used in that ((Proc.Natl.Acad.Sci.USA 74 (1977), 5463-5467)) dna sequencing for Sanger according to Sanger.Except that these enzymes, there are a large amount of 37 ℃ of archaeal dna polymerases with different qualities more, these enzymes can use in the methods of the invention.
The most common and thermostable DNA polymerases is the Taq archaeal dna polymerase that comes from thermus aquaticus (Thermusaquaticus), and this enzyme optimum temperuture is 75 ℃, 95 ℃ very stable, this enzyme obtains commercial.The Taq archaeal dna polymerase is 5 '-a 3 ' archaeal dna polymerase of height persistence, and this enzyme does not have 3 '-5 ' exonuclease activity.It is usually used in Standard PC R, sequencing reaction and mutagenesis PCR (Cadwell und Joyce (PCR Methods Appl.3 (1994), 136-140, Agrogoni and Kaminski (Methods Mol.Biol.23 (1993), 109-114)).Come from the Tth archaeal dna polymerase of thermus thermophilus (Thermus thermophilus) HB8 and come from the dwell Tfl archaeal dna polymerase of hot bacterium (Thermus flavas) of yellow and have similar characteristic.Yet (Cusi etc. (Biotechniques 17 (1994), 1034-1036)), Tth archaeal dna polymerase have intrinsic reversed transcriptive enzyme (RT) activity again in the presence of magnesium ion.In addition, many have 3 ', but the heat-stable DNA polymerase that does not have 5 ' exonuclease activity is commercial on sale: the Pwo archaeal dna polymerase of 5 Si Shi fireball bacterium (Pyrococcus woesei), the Tli of Thermococcus litoralix, Vent and DeepVent archaeal dna polymerase, Pfx and the Pfu archaeal dna polymerase of fierce fireball bacterium (Pyrococcus furiosus), generally the dwell Tub archaeal dna polymerase of hot bacterium (Thermus ubiquitous), the Tma of Thermotoga maritima (Thermotoga maritime) and UlTma archaeal dna polymerase (Newton and Graham, PCR, Spektrum Akad.Verlag Heidelberg (1994), 1)).Using the polysaccharase that lacks 3 ' calibration nucleic acid 5 prime excision enzyme activity is for the wrong the least possible PCR product that increases.At last, the archaeal dna polymerase that 5 '-3 ' and 3 '-5 ' exonuclease activity all lacks can obtain with the segmental form of Stoffel of Taq archaeal dna polymerase, Vent-(exo-) archaeal dna polymerase and Tsp archaeal dna polymerase.In the archaeal dna polymerase (reversed transcriptive enzyme) that RNA relies on, modal enzyme comprises the AMV reversed transcriptive enzyme of avian myeloblastosis virus, the M-MuLV reversed transcriptive enzyme of Moloney murine leukemia virus and human immunodeficiency virus's hiv reverse transcriptase, many manufacturers also sell these enzymes, for example NEB, LifeTechnologies, Quantum Biotechnologies.As hiv reverse transcriptase, the AMV reversed transcriptive enzyme has relevant RNase H activity, and this activity significantly reduces in the M-MuLV reversed transcriptive enzyme.M-MuLV and AMV reversed transcriptive enzyme all lack 3 '-5 ' exonuclease activity.
In the RNA polymerase that DNA relies on, modal enzyme comprises the e. coli rna polysaccharase, come from Salmonella typhimurium (Salmonella thyphimurium) LT2 that phage SP6 infects the SP6 RNA polymerase, come from the T3 RNA polymerase of phage T3 and come from the T7 RNA polymerase of phage t7.
In the inventive method embodiment preferred, the template strand in present method step (f) is a dna molecular, and the archaeal dna polymerase that relies on of DNA to be used for template guided strand synthetic.
In particularly preferred embodiments, used herein is non-thermostable DNA polymerases, particularly preferably is the enzyme with 5 '-3 ' and 3 '-5 ' exonuclease activity, for example Escherichia coli polymerase I.
As alternative, also can use the non-heat-stable DNA polymerase that has 3 '-5 ' exonuclease activity but do not have 5 '-3 ' exonuclease activity, for example the T4 archaeal dna polymerase of the T7 archaeal dna polymerase of the Klenow fragment of e. coli dna polymerase I, phage t7 or phage T4.
In addition, also can use the non-heat-stable DNA polymerase that had not both had 3 '-5 ' exonuclease activity, also do not had 5 '-3 ' exonuclease activity, for example 3 '-5 '-exo (-) variant of the Klenow fragment of e. coli dna polymerase I.
In addition, particularly preferred embodiment is used heat-stabilised poly synthase (for example Taq polysaccharase, Pwo polysaccharase), these enzymes have 5 '-3 ' and 3 '-5 ' exonuclease activity simultaneously, or have 5 ' exonuclease activity but do not have 3 ' exonuclease activity, the Tfl archaeal dna polymerase of the Tth archaeal dna polymerase of the Taq archaeal dna polymerase of thermus aquaticus, thermus thermophilus HB8 or the yellow hot bacterium that dwells for example.
As alternative, heat-stable DNA polymerase can have 3 '-5 ' but do not have 5 '-3 ' exonuclease activity, as, for example come from VentR archaeal dna polymerase, the Pwo archaeal dna polymerase of 5 Si Shi fireball bacterium, the DeepVentR archaeal dna polymerase that comes from Thermococcus litoralis or Tli archaeal dna polymerase, the Pfu archaeal dna polymerase that comes from fierce fireball bacterium or Pfx archaeal dna polymerase or come from Tma archaeal dna polymerase or the UITma archaeal dna polymerase of Thermotoga maritima.
Can use does not further have the heat-stabilised poly of 3 '-5 ' and 5 '-3 ' exonuclease activity synthase yet, for example comes from exo (-) variant of the Stoffel fragment of the Taq archaeal dna polymerase of thermus aquaticus, the Tsp archaeal dna polymerase that comes from Thermococcus litoralis or VentR archaeal dna polymerase or DeepVentR archaeal dna polymerase.
If use the heat-stabilised poly synthase, after polymeric enzyme reaction preferably is right after step (e), need not carry out intermediary purifying or further sample preparation.
In another preferred embodiment of the inventive method, carrying out the template strand of template guided strand in synthetic with the step (f) of the inventive method is the RNA molecule.In this case, the archaeal dna polymerase that the synthetic RNA of use of template guided strand relies on, preferably come from avian myeloblastosis virus the AMV reversed transcriptive enzyme, come from human immunodeficiency virus's hiv reverse transcriptase or come from the M-MuLV reversed transcriptive enzyme of Moloney murine leukemia virus.Further preferably use thermostable reverse transcriptases, very specifically come from the Tth archaeal dna polymerase of thermus thermophilus, this enzyme has the intrinsic reverse transcriptase activity.
Embodiment 1
Unless have illustratedly in other place, experimental basis Current Protocols in MolecularBiology carries out.
I., initial substance is provided
4 kinds of lipase Variants are as the LipAL167A of LipA F142W, the pBP2007 coding of LipA S87T, the pBP2006 coding of LipA H86W, the pBP2008 coding of initial substance: pBP2035 coding.
1. the plasmid prepared product of following plasmid:
| Plasmid | Carrier | The carrier size | Insert fragment | Insert clip size | Enzyme variants |
| ?pBP2006 | ?pBSIIKS | 2961bp | 56-20 | 1142bp | F142W |
| ?pBP2007 | ?pBSIIKS | 2961bp | 98-10 | 1142bp | L167A |
| ?pBP2008 | ?pBSIIKS | 2961bp | 124-9 | 1142bp | S87T |
| ?pBP2035 | ?pBSIIKS | 2961bp | 198-1-3 | 1142bp | H86W |
2. with restriction enzyme HindIII and SacI cutting plasmid.
3. use GFX test kit (Pharmacia), separate by gel extraction and insert fragment.
4. resuspended dna fragmentation in water.
5. DNA concentration is adjusted to～250ng/ μ l.
II. the generation of strand breach and de novo synthesis
1. use nick translation test kit (Roche, catalog number (Cat.No.) 976776), be used to insert 4 independent reaction mixtures that fragment 56-20,98-10,124-9 and 198-1-3 carry out nick translation.
| Component | Volume |
| Insert fragment concentrations and be～dna solution of 250ng/ μ l | ?16μl |
| ?dATP?0.4mM | ?2.5μl |
| ?dCTP?0.4mM | ?2.5μl |
| ?dGTP?0.4mM | ?2.5μl |
| ?dTTP?0.4mM | ?2.5μl |
| 10 * damping fluid | ?5μl |
| Water | ?14μl |
| Enzyme mixture 1) | ?5μl |
| Cumulative volume | ?50μl |
1)Enzyme mixture:
Archaeal dna polymerase and DNAseI in 50% (v/v) glycerine.
2. mixture was hatched 90 minutes at 15 ℃.
3. by adding 5 μ l 0.5M EDTA, pH8.8 and termination reaction.
4. precipitation, washing, drying, and mixture is resuspended in the 20 μ l water.
III. there is not primer PCR
The PCR system (Roche, catalog number (Cat.No.) 2140306) that GC is rich in use carries out PCR to reaction mixture.
| Component | Volume |
| 6 μ l mixtures of every kind of mixture among the II.4 | 24μl |
| dNTP?10mM | 1μl |
| Be rich in the resolution solution 5M of GC | 10μl |
| Be rich in the reaction buffer of GC | 10μl |
| PCR level water | 4μl |
| Be rich in the enzyme mixture of GC 2) | 1μl |
| Cumulative volume | 50μl |
2)Stable Taq archaeal dna polymerase and Tgo archaeal dna polymerase.
The PCR condition:
| Temperature | Time | Cycle number |
| ????95℃ | 5 minutes | |
| ????55℃ | 60 seconds | ????45 |
| ????72℃ | 60 seconds | |
| ????95℃ | 30 seconds | |
| ????72℃ | 10 minutes | |
| ????4℃ | X minute |
IV., primer PCR is arranged
The PCR system (Roche, catalog number (Cat.No.) 2140306) that GC is rich in use carries out PCR to reaction mixture:
| Component | Volume |
| The PCR product of step 3 | 5μl |
| dNTP?10mM | 1μl |
| Primer MAT16*6.25 μ M | 2μl |
| Primer MAT19*6.25 μ M | 2μl |
| Be rich in the resolution solution 5M of GC | 10μl |
| Be rich in the reaction buffer of GC | 10μl |
| PCR level water | 19μl |
| Be rich in the enzyme mixture of GC | 1μl |
| Cumulative volume | 50μl |
* primer MAT16=5 '-GATCGACGTAAGCTTTAACGATGGAGAT-3 '
* primer MAT19=5 '-CATCGGGCGAGCTCCCAGCCCGCCGCG-3 '
The PCR condition:
| Temperature | Time | Cycle number |
| ????95℃ | 5 minutes | |
| ????52℃ | 30 seconds | ????25 |
| ????72℃ | 60 seconds | |
| ????95℃ | 30 seconds | |
| ????72℃ | 10 minutes | |
| ????4℃ | X minute |
V. segmental clone and analysis
1.20 the PCR product in the μ l step 4 cuts with HindIII and SacI.
2. precipitation, washing, dry and with the resuspended mixture of 20 μ l water.
3. use GFX test kit (Pharmacia) purifying cutting fragment.
4. fragment is connected with the carrier pBSIIKS (Stratagene) with HindIII and SacI cutting in advance.
5. will connect mixture transformed into escherichia coli XL1-Blue.
6. has the sequential analysis of inserting segmental 29 clones.
Claims (11)
1. produce the method with the polynucleotide molecule that changes characteristic, it comprises and carries out the circulation that at least one comprises the steps:
(a) provide a kind of double-stranded polynucleotide molecule or the colony of double-stranded polynucleotide molecule is provided, independent polynucleotide have at least one homologous sequence part and at least one heterologous sequence part in the wherein said colony,
(b) in described double-stranded polynucleotide molecule, produce the strand breach,
(c) begin to carry out 5 '-3 ' nuclease degradation from described strand breach, 3 ' end at described strand breach carries out 5 '-3 ' de novo synthesis and mobile simultaneously,
(d) preparation strand polynucleotide molecule,
(e) the partially double stranded polynucleotide molecule of the strand polynucleotide molecule that provides of preparation process (d),
(f) initial to carry out template guided nucleic acid synthetic from the partially double stranded polynucleotide molecule of step (e) preparation,
Can be successively or implementation step (b) and (c) simultaneously.
2. the described method of claim 1 wherein carries out comprising more than one the circulation of step (a)-(f).
3. the described method of claim 2 wherein selects step to carry out after one, several or all circulations, the genotype of described selection step and polynucleotide or with its phenotype or all relevant with its genotype and phenotype.
4. any described method among the claim 1-3, wherein sequence-specific nickase is introduced described strand breach in step (b).
5. any described method among the claim 1-3, wherein non-sequence-specific nickase is introduced described strand breach in step (b).
6. any described method among the claim 1-5, wherein dna polymerase i uses in step (c).
7. any described method among the claim 1-6 is wherein passed through to select appropriate reaction conditions, and the error rate of described de novo synthesis increases in step (c).
8. any described method among the claim 1-6 is wherein passed through one or more excessive ribonucleoside triphosphotes, and the error rate of described de novo synthesis increases.
9. any described method among the claim 1-6 is wherein passed through to use one or more nucleotide analogs, and the error rate of described de novo synthesis increases.
10. any described method among the claim 1-6 is wherein passed through to change salt concn, and the error rate of described de novo synthesis increases.
11. any described method among the claim 1-6, wherein by having the polysaccharase that reduces or do not have proofreading activity, the error rate of described de novo synthesis increases.
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|---|---|---|---|
| DE10223057A DE10223057A1 (en) | 2002-05-24 | 2002-05-24 | Process for the production of polynucleotide molecules |
| DE10223057.9 | 2002-05-24 |
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|---|---|
| CN1656221A true CN1656221A (en) | 2005-08-17 |
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|---|---|---|---|
| CNA038119277A Pending CN1656221A (en) | 2002-05-24 | 2003-05-21 | Method for creating polynucleotide molecules |
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| EP (1) | EP1511842A2 (en) |
| JP (1) | JP2005529596A (en) |
| KR (1) | KR20050004207A (en) |
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| AU (1) | AU2003240683A1 (en) |
| CA (1) | CA2485218A1 (en) |
| DE (1) | DE10223057A1 (en) |
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| GB2574197B (en) * | 2018-05-23 | 2022-01-05 | Oxford Nanopore Tech Ltd | Double stranded polynucleotide synthesis method and system. |
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| CA2422749A1 (en) * | 2000-09-21 | 2002-03-28 | Merck & Co., Inc. | Method for generating recombinant polynucleotides |
| WO2002079468A2 (en) * | 2001-02-02 | 2002-10-10 | Large Scale Biology Corporation | A method of increasing complementarity in a heteroduplex polynucleotide |
-
2002
- 2002-05-24 DE DE10223057A patent/DE10223057A1/en not_active Withdrawn
-
2003
- 2003-05-21 AU AU2003240683A patent/AU2003240683A1/en not_active Abandoned
- 2003-05-21 MX MXPA04011554A patent/MXPA04011554A/en not_active Application Discontinuation
- 2003-05-21 CA CA002485218A patent/CA2485218A1/en not_active Abandoned
- 2003-05-21 WO PCT/EP2003/005308 patent/WO2003100058A2/en not_active Application Discontinuation
- 2003-05-21 EP EP03730082A patent/EP1511842A2/en not_active Withdrawn
- 2003-05-21 KR KR10-2004-7018944A patent/KR20050004207A/en not_active Application Discontinuation
- 2003-05-21 CN CNA038119277A patent/CN1656221A/en active Pending
- 2003-05-21 JP JP2004508297A patent/JP2005529596A/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| DE10223057A1 (en) | 2003-12-11 |
| JP2005529596A (en) | 2005-10-06 |
| KR20050004207A (en) | 2005-01-12 |
| WO2003100058A3 (en) | 2004-09-02 |
| EP1511842A2 (en) | 2005-03-09 |
| MXPA04011554A (en) | 2005-03-07 |
| AU2003240683A1 (en) | 2003-12-12 |
| CA2485218A1 (en) | 2003-12-04 |
| WO2003100058A2 (en) | 2003-12-04 |
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