WO1998005765A1 - Adn double brin a extremite(s) cohesive(s) et procede de rearrangement d'adn y recourant - Google Patents

Adn double brin a extremite(s) cohesive(s) et procede de rearrangement d'adn y recourant Download PDF

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Publication number
WO1998005765A1
WO1998005765A1 PCT/DK1997/000317 DK9700317W WO9805765A1 WO 1998005765 A1 WO1998005765 A1 WO 1998005765A1 DK 9700317 W DK9700317 W DK 9700317W WO 9805765 A1 WO9805765 A1 WO 9805765A1
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dna
gene
sequence
stranded
double
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PCT/DK1997/000317
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English (en)
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Yoshiaki Miyota
Shiro Fukuyama
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Novo Nordisk A/S
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Priority to AU36917/97A priority Critical patent/AU3691797A/en
Publication of WO1998005765A1 publication Critical patent/WO1998005765A1/fr

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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
    • 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
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules

Definitions

  • the present invention relates to a double-stranded DNA with a cohesive end or cohesive ends having a desired sequence and a method for producing it, and also a method for shuffling a DNA using DNA blocks with a cohesive end or cohesive ends, the DNA as shuffled according to the method, a DNA pool to be obtained according to the shuffling method, and also a genetic product to be produced by the use of the DNA pool .
  • 095/22625 disclosed is a method for forming novel genes by dividing a plurality of genes at random and homolo- gously reco bining them to reconstruct novel genes.
  • this is one method for forming chimera genes.
  • the genes to be formed by this method are similar to the original genes, and the former shall have the essential base sequences of the latter.
  • it is difficult to desire the impartation of some additional functions to organisms which they could not gain during the steps of their evolution.
  • One method for this may be to prepare a nucleic acid pool that covers all base combinations.
  • the total number of the base sequences that may code for a relatively small protein with 100 amino acids (300 bp) is an enormous number of 4 300 (about 10 180 ) , and it is in fact impossible to prepare the nucleic acid pool that may cover all of them.
  • restriction enzymes it is possible to prepare a nucleic acid pool including various molecules by blending several kinds of DNA blocks having the same cohesive end or blunt end followed by ligating them at random, and to select from this pool some molecules having desired properties. According to this method, however, the DNAs to be used must have prede- termined restriction enzyme recognizing sites. Even though the DNAs have such restriction enzyme recognizing sites, there is an extremely small probability that the sites are positioned at the desired sites. In this method, in addition, the both ends of the blocks must be of the same type, and there is a high probability that the blocks are therefore self-ligated. A means of forming the restriction enzyme recognizing sites through site-specific mutation may be taken in order to overcome these problems.
  • the matter as to whether or not the blocks can be ligated in accordance with the predetermined frame is likely much governed by chance. That is, the matter as to whether or not a desired protein can be produced without misreading the reading frame of the codon shall be governed by chance. Therefore, the method is extremely inefficient.
  • the subject matter of the present invention is to provide a method for efficiently obtaining base sequences that exist in spaces greatly different from those of naturally-existing base sequences, and also to provide genetic products to be obtained by expressing, as genes, the nucleic acid sequences that are obtained in that manner and that do not exist naturally.
  • the sequence space of a gene includes the full-length sequence thereof to be theoretically constituted by a combination of four bases, A, G, C and T.
  • a base sequen- ce that codes for a protein composed of a number "n" of amino acids shall be constructed by selecting and sequencing any desired one of the four bases for a total of 3n-times, therefore including 4 3n combinations.
  • a protein composed of 100 amino acids shall include different base sequences of about 10 18 types as so mentioned hereinabove.
  • sequencing spaces for proteins shall extend unlimitedly. During the steps of evolution of organisms, only a part of such sequencing spaces have been examined, and there is a great probability that some sequences coding for proteins which may have some extremely excellent functions could exist in the other great sequencing spaces .
  • the protein engineering studies which have been and are being made in many laboratories and institutes at present are essentially directed to the creation of novel proteins having functions superior to those of naturally-existing proteins, and one essential approach made therein to this pur- pose is to substitute amino acids in existing sequences, as so mentioned hereinabove.
  • amino acid substitution is nothing but the essential means that organisms have carried out during the steps of their evolution or, that is, such is the imitation of organisms and is to search only around the sequences that organisms already examined. In addition, there is a probability that the sequences thus obtained will be those that were already weeded out in the past.
  • the present inventors have considered that, in order to be greatly apart from the sequencing spaces that organisms already examined, if we carry out such matters that could not have been carried out by organisms, the purpose will be attained.
  • the present invention provides the following:
  • a DNA with a cohesive end comprising (a) a double- stranded DNA having the same sequence as that of a part of a gene, and (b) a single-stranded DNA having a base sequence that exists on said gene at the site not adjoining the part corresponding to said double-stranded DNA or a base sequence which said gene does not have, wherein the single-stranded DNA is linked to either one end of the double-stranded DNA to form a cohesive end.
  • a DNA with cohesive ends comprising (a) a double- stranded DNA having the same sequence as that of a part of a gene, (b) a first, single-stranded DNA having a base sequence that exists on said gene at the site not adjoining the part corresponding to said double-stranded DNA or a base sequence which said gene does not have, and (c) a second, single- stranded DNA having a base sequence that exists on said gene at the site adjoining the part corresponding to said double- stranded DNA, wherein the second, single-stranded DNA is linked to said double-stranded DNA at one end corresponding to said adjoining site, while the first, single-stranded DNA is linked thereto at the other end of the complementary strand opposite to said end, thereby forming cohesive ends.
  • a method for producing a DNA with a cohesive end or cohesive ends wherein a part of a DNA, as a template, and an oligonucleotide containing at least one ribonucleotide, as a primer, are subjected to DNA polymerase reaction to prepare a double-stranded DNA, then the ribonucleotide (s) is/are removed through enzymatic reaction or chemical reaction, and the nu- cleotide(s) remaining at the 5 ' -terminal (s) of the site(s) at which said ribonucleotide (s) existed are removed.
  • a method for producing the DNA with a cohesive end of the previous 1) comprising the following steps a) to d) : a) a step of linking (i) an oligonucleotide having the same base sequence as that of a part of a gene DNA to (ii) an oligonucleotide having a base sequence that exists on the gene at the site not adjoining the base sequence of (i) or a base sequence which the gene does not have, and containing at least one ribonucleotide, in such a manner that the oligonucleotide (ii) is positioned at the 5 '-terminal of the oligonucleotide
  • a method for producing the DNA with cohesive ends of the previous 2) comprising the following steps a) to d) : a) a step of linking (i) an oligonucleotide having the same base sequence as that of a part of a gene DNA to (ii) an oligonucleotide having a base sequence that exists on the gene at the site not adjoining the base sequence of (i) or a base sequence which the gene does not have, and containing at least one ribonucleotide, in such a manner that the oligonucleotide (ii) is positioned at the 5 '-terminal of the oligonucleotide (i); b) a step of preparing a double-stranded DNA through DNA polymerase reaction between a DNA containing the part corresponding to the oligonucleotide (i) in said a) , as a template, and (i) the linked oligonucleotide as obtained in the previous step a)
  • a method for shuffling a DNA comprising dividing a DNA into a plurality of DNA blocks each having a cohesive end or cohesive ends, followed by ligating them together into a sequence that is different from the sequence of the original, non-divided DNA.
  • a method for shuffling a DNA comprising applying the method of any one of the previous 5) to 7) to various sites of a DNA, thereby dividing the DNA into a plurality of DNA blocks each having a cohesive end or cohesive ends, at least one block of which shall have a cohesive end that is complementary to the cohesive end of another block not having been directly adjacent to said one block on the original DNA, followed by ligating them together into a sequence that is different from the sequence of the original, non-divided DNA.
  • a method for producing a DNA pool comprising applying the method of any one of the previous 5) to 7) to various sites of a template DNA to thereby prepare a mixture of DNA blocks each having a cohesive end or cohesive ends that satisfies the following conditions, followed by ligating these into any desired sequences:
  • Each block has a double-stranded site having the same sequence as that of a part of the template DNA.
  • Condition 2 At least two of the blocks that constitute the block mixture further have, in addition to said double- stranded site, s single-stranded site (cohesive end) that is complementary to the cohesive end of blocks that are not directly adjacent to said blocks on the template DNA.
  • Condition 3 The block mixture contains at least two different blocks which are the same in the double-stranded site but are different only in the single-stranded site and which satisfy the condition 2. 21) The method for producing a DNA pool according to the previous 20) , wherein the template DNA is a gene that codes for an enzymatic function or a control gene DNA for the gene.
  • the template DNA is a gene DNA that codes for any one of proteases, lipases, cellulases, amylases, catalases, xylanases, oxidases, dehydrogenases, oxygenases and reductases.
  • DNA with Cohesive End(s) The present invention provides a DNA with any desired cohesive end or ends (herein referred to as "DNA with cohesive end(s)" unless otherwise specifically indicated).
  • the cohesive end as referred to herein indicates a single-stranded site as protruded from the end of a double-stranded DNA.
  • Such a cohesive end may be formed when a DNA is cleaved with a restriction enzyme such as EcoRI .
  • the base sequence of the thus-formed cohesive end is defined, depending on the restriction enzyme used, and its length is generally composed of several bases or so.
  • the sequence of the resulting double-stranded part of the DNA is also limited to the region as sandwiched between the restriction enzyme recognizing sites.
  • the DNA with cohesive end(s) of the present invention may have a structure in which a cohesive end or cohesive ends having a desired length and a desired sequence is/are added to the end or ends of a double-stranded DNA having a desired sequence.
  • sequence of the double-stranded part of the DNA with cohesive end(s) of the present invention is not specifically defined.
  • the sequence may be the same as that of a part of a gene.
  • its length may be generally composed of 50 base pairs (bp) or more, preferably 45 bp or more.
  • the sequence of the cohesive end is not also specifically defined, but in order to prevent the self-ligation thereof in various reactions, it is preferable that the sequence does not form a stem structure.
  • the "sequence to form a stem structure" as referred to herein includes, for example, AATT, which shall have just the same sequence as that of its complementary strand (TTAA) .
  • the length of the cohesive end may be generally 2 bp or more, preferably from 15 bp to 30 bp. If the cohesive end is too long, it may form a secondary struc- ture whereby the intermolecular annealing will be difficult. However, if it is too short, its melting temperature (Tm) is low and the annealing will be unstable.
  • the cohesive end may be linked to either the 3 '-terminal or the 5 '-terminal of the double-stranded DNA, but is prefe- rably linked to the 3 '-terminal thereof.
  • the cohesive end may be linked to either only one terminal of the double-stranded DNA or the both terminals thereof.
  • the DNA with cohesive end(s) of the present invention can be produced typically according to a method comprising the following steps a) to d) .
  • the method mentioned below is directed to the production of a DNA with a cohesive end, which has a structure to be represented by a formula (2) :
  • oligonucleotide, a which is complementary to its terminal, X
  • an oligonucleotide, c having the same sequence as that of the other terminal, c, are prepared.
  • X and c each may have a sequence having a base length of from 15 to 30 bp or so.
  • oligonucleotide b, which is complementary to the sequence to be prepared by removing one base (this is referred to as X) from the 5'- terminal of the sequence of the intended cohesive end, a " .
  • the base sequence, & may be a part of the above-mentioned DNA or may be any other sequence that the DNA does not have.
  • oligonucleotides may be prepared by any methods. If their sequences are previously known, they can be synthesized, using a known DNA synthesizer.
  • oligonucleotides a and b
  • a ribonucleotide is linked together via a ribonucleotide.
  • This linkage can be attained by ordinary synthesizing methods. For example, it can be attained according to the process mentioned below.
  • a phosphoryl group is added to the 5 ' -terminal of the oligonucleotide, a, according to the reaction of the following formula (3) : (3) wherein (P) is a phosphoryl group.
  • This reaction can be effected by the action of a polynucleotide kinase.
  • ATP is used in an amount of from 2 to 10 times or so, by mol, relative to the oligonucleotide, a.
  • the reaction temperature may be from 30 to 40°C or so.
  • the reaction time may be from 10 minutes to 1 hour or so. Most suitably, the pH is from 7 to 9 or so.
  • the oligonucleotide is represented by a ' .
  • a ribonucleotide is added to the 3'- terminal of the oligonucleotide, b, according to the reaction of the following formula (4) : (4) wherein X is any one of ATP, GTP, CTP and UTP; (rX) is a ribonucleotide.
  • This reaction can be effected by the action of, for example, a terminal deoxynucleotidyl transferase.
  • XTP nucleoside triphosphate
  • a-1 a ribonucleotide that corresponds to the base X in the previous step (a-1) .
  • the nucleoside triphosphate is used in an amount of from 2 to 10 times, by mol, relative to the oligonucleotide, b.
  • the reaction temperature may be from 30 to 40°C or so.
  • the reaction time may be from 30 minutes to 2 hours or so.
  • the oligonucleotide is represented by b'.
  • the sequence of b' is complementary to the sequence, -s ⁇ .
  • This reaction can be effected by the action of an RNA ligase in the presence of ATP and divalent metal ions (Japanese Patent Application Laid-Open No. 5-292967) .
  • Divalent metal ions useful in this reaction include, for example, magnesium ions and manganese ions, but preferred are magnesium ions.
  • the ligase employable is an RNA ligase.
  • the RNA ligase is an enzyme to catalyze the ligation of the hydroxyl group at the 3 ' -terminal and the phosphoryl group at the 5 ' -terminal , and this also efficiently catalyzes the ligation of a polydeoxyribonucleotide having a ribonucleotide only at its 3'- terminal and a polydeoxyribonucleotide with a 5 '-terminal phosphoryl group.
  • a T4 RNA ligase is effected in a buffer, at a pH of from 7 to 9 and at a temperature of from 10 to 40°C, over a period of from 30 to 180 minutes.
  • the oligonucleotides may be reacted in a solution comprising 50 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 0.1 mM ATP, 10 mg/ liter BSA, 1 mM hexaammine cobalt chloride (HCC) and 25 % polyethylene glycol 6000, at 25°C for 60 minutes or longer.
  • a double-stranded DNA containing the sequence, X, as prepared in the previous step (a-1) , as a template, and using the linked oligonucleotide, b'-a', as prepared in the previous step (a-2), as a primer, prepared is a double-stranded DNA through DNA polymerase reaction.
  • a double-stranded DNA containing the sequence, X, and a sequence, 3, on their strands is subjected to thermal or alkaline denaturation to give single-stranded DNAs, which are added to the primer of b'-a" and subjected to PCR with the oligonucleotide, c, as prepared in the previous step (a-1) .
  • the primer annealing condition and the polymerase reaction condition to be employed herein may be the same as those in ordinary polymerase reaction.
  • the DNA polymerase to be employed herein may be any and every enzyme that can catalyze the DNA chain-extending reaction, which includes, for example, Taq polymerase, Klenow fragment, DNA polymerase I, etc. As a result of this reaction, obtained is a double-stranded DNA with blunt ends, which is represented by the a formula (6) :
  • Step c) the ribonucleotide is removed from the double- stranded DNA through enzymatic reaction or chemical reaction.
  • One example of useful enzymes is a ribonuclease.
  • the reaction is generally effected at a pH of from 6 to 8 or so, at from 30 to 70°C or so, over a period of from 10 to 60 minutes or so.
  • non-enzymatic chemicals usable herein mentioned are sodium hydroxide and the like.
  • a partly-discontinuous, double-stranded DNA of the following formula (7) in which the part corresponding to the above- mentioned base, X, has been deleted.
  • the nucleotide existing at the 5'- terminal of the above-mentioned deletion is removed.
  • the double-stranded DNA, from which the ribonucleotide has been removed in the previous step c) is heated at from 50 to 90°C or so.
  • the polynucleotide that has been separated from the strand through this reaction can be removed, using, for example, a span column or the like.
  • a desired sequence, ⁇ t ⁇ t which does not adjoin the sequence, X, in the template DNA was introduced into the DNA to form the cohesive end.
  • an oligonucleotide, c' which is different from the oligonucleotide, c, in that its 3 '-terminal deoxyribonucleotide is substituted with a ribonucleotide, may be used as the primer in place of the oligonucleotide, c, to prepare a double-stranded DNA with two cohesive ends of a formula (8) : (8).
  • the present invention also provides a method of shuffling a DNA, which is characterized by using DNAs with cohesive end(s) .
  • shuffling as referred to herein in- dicates the operation of dividing a DNA into plural blocks followed by re-sequencing them into a desired, different sequence.
  • one DNA has a sequence composed of a number, n, of blocks, as represented by a formula (9) : A - al - a2 - . . . . - a n - B (9) wherein the starting end A and/or the terminal end B may be omitted
  • this may be shuffled according to the present invention to give a different DNA to be represented by a formula (10) : A - al' - a2' - . . . . - a x - B (10) wherein al • , a2 * , . . . , ax are blocks that are independently selected from the group of al, a2, . . .
  • the total number of the blocks al', a2 ' , . . . , a x may not be the same as the total number of the blocks cii f SL f * • • • f £t_n •
  • Fig. 1 The principle of the DNA shuffling of the present invention which utilizes DNAs with cohesive end(s) is graphically illustrated in Fig. 1.
  • the DNA is shuffled at the intermediate part, pi - p2 - p3 (the uppermost row) into p3 - pi - p2 (the lowermost row) , without changing the both ends PA and PB-
  • This shuffling operation is useful as a method for obtaining gene sequences that have not heretofore existed naturally, without changing the sequences of the promoter and the terminator.
  • the above-mentioned method of preparing DNAs with cohesive end(s) is applied first to the parts PA, pi, p2, p3 and p ⁇ constituting the template DNA, to thereby prepare DNA blocks, al, a2 and a3, each having the structure with two cohesive ends (formula (8)), and DNA blocks, A and B, each having the structure with one cohesive end (formula (2)).
  • the cohesive ends, a A , aif, a 2 f and a3 f are formed by removing the corresponding complementary strand from the blocks, PA, pi/ p2 and p3, respectively.
  • the cohesive ends, a ⁇ r , a2r, a 3r an( * a B, are designed according to the desired sequence to be prepared after the shuffling.
  • the end, a ⁇ r is designed to be a complementary strand to the end, a3f, and the block, al is ligated to the block a3 after the shuffling.
  • the ligation is conducted, using a DNA ligase in the presence of ATP.
  • the type of the DNA ligase to be employed herein is not specifically defined. In this embodiment, since the single- stranded part of each cohesive end is long, it is unnecessary to employ the ordinary reaction at 16°C, but a thermophilic DNA ligase is advantageously employed.
  • a2r; a 3r an d a B are designed to be the complementary strands to a ⁇ _f, aA and a2f, respectively, in the same manner as above.
  • a sequence having a structure of A - a3 - al - a2 - B is finally obtained.
  • This is seemingly the same as the re- sequenced order of P - p3 - pi - p2 - PB to be obtained by dividing the original DNA into the constitutive blocks pi, p2, p3 , PA and PB, followed by re-sequencing these into a different sequence.
  • any other desired sequences can be realized in the same manner as above. If the block, A or B, is made to have two cohesive ends, while the other blocks are made to have one cohesive end, it is possible to obtain still different sequences through shuffling where the latter blocks with one cohesive end are positioned at the terminals.
  • the shuffling of the invention it is also possible to introduce foreign DNA block(s) with cohesive end(s), which are not in the original gene, into the gene DNA.
  • the terminal of one gene for example, the block A in the above-mentioned embodiment, may be processed into a DNA block with two cohesive ends, if desired.
  • the blocks which are the units to be shuffled, are oligonucleotides or polynucleotides composed of 2 or more nucleotides (hereinafter referred to as "oligonucleotides") . In general, these are preferably composed of 30 or more nucleotide units, more preferably 45 or more nucleotide units.
  • the uppermost limit of the block length is not specifically defined, provided that the block length is shorter than the length of one gene. If, however, the block length is too large, the re-sequenced DNA to be obtained by the shuffling shall have many non-mutated base sequence parts. Therefore, in general, the block length is preferably within the range of from 10 to 35 % of the length of a gene.
  • the gene to be shuffled is a gene that codes for a protein
  • the gene blocks, oligonucleotides have the same reading frame before and after the division.
  • the gene blocks to be shuffled are desirably so designed that they are translated to always give the corresponding amino acid sequences, irrespective of their relative positions in the shuffled sequence.
  • the double-stranded parts and the cohesive ends shall be selected for their codon units in accordance with the reading frame of the gene DNA to be shuffled. Needless-to-say, it is unnecessary to conduct the division into segment blocks with genetic meanings.
  • the kind of the gene to be shuffled according to the present invention is not specifically defined.
  • Employable herein is any and every gene that is composed of polynucleotide chains and contains a coding region necessary for expressing a protein or RNA.
  • the nucleotide unit may contain any molecule of deoxy- ribonucleotides or ribonucleotides.
  • genes coding for proteins especially enzymes, or control genes for enzymatic functions.
  • enzymes include proteases, lipa- ses, cellulases, amylases, catalases, xylanases, oxidases, dehydrogenases, oxygenases and reductases.
  • the kind of the gene to which the present invention is directed is not specifically defined but shall be such that, when it is introduced into a suitable host, the host can produce the genetic product through expression of the gene.
  • genes as cloned from living organisms include artificially synthesized genes, and even genes as cloned from living organisms and artificially mutated.
  • prokaryotes with definite enzyme producibility .
  • prokaryotes mentioned are bacillus bacteria.
  • One example of the genes derived from such bacteria is a protease API21 gene derived from Bacillus NKS-21 (FERM BP-93-1) (Japanese Patent Application Laid-Open No. 5-91876, Sequence Number 1).
  • the present invention also provides a DNA pool to be obtained according to the above-mentioned shuffling method.
  • the "DNA pool” as referred to herein means a high-density mixture of two or more DNAs.
  • the DNA pool of the present invention can contain a particular number or more, for example, 10 or more different DNA molecules having different structures. It is desirable that, when the mixture, DNA pool is directly used in biochemical operation or reaction, it is in such a form that all the plural nucleic acid components constituting it can be reacted. However, the form of the mixture, DNA pool is not specifically defined, and the DNA pool may be either in solution or dry mixture.
  • a plurality of cohesive ends for each block are prepared in the above-men- tioned shuffling process.
  • complementary strands to the other cohesive ends, aA and a2f are prepared in addition to the complementary strand to a3f, then DNAs of A - al - a2 - B and A - al -a2 - al can be ob- tained.
  • a complementary strand to the other cohesive end alf of al is added, it is also possible to produce other DNAs comprising a series of the same blocks, such as A - al - al - al.
  • a DNA is divided into blocks of al, a2 , a3 , . . . , an- Then, each block is processed to have a cohesive end or cohesive ends according to the above-mentioned process.
  • the cohesive ends are designed to be oligonucleotides that are complementary to the cohesive ends of the other blocks or are complementary to the other cohesive end of themselves. All or a part of the thus-obtained DNA blocks are mixed and ligated to each other, thereby producing a nucleic acid pool containing different nucleic acids composed of the blocks as differently sequenced at random.
  • the thus-shuffled, single or mixed, double-stranded DNAs are blunted.
  • the blunting may be omitted, if DNA blocks with one cohesive end are positioned at the ends of the shuffled, double-stranded DNA.
  • the 5 '-terminal of the sequence containing a DNA block with a predetermined promoter sequence which is based on the direction of the promoter, is not made to have a cohesive end but is made to have a blunt end
  • the 3 '-terminal of the sequence containing a DNA block with a predetermined terminator sequence which is based on the direction of the terminator, is not made to have a cohesive end but is also made to have a blunt end.
  • the thus-shuffled DNA is inserted into a desired vector, preferably an expression vector such as pKK223-3, using a DNA ligase.
  • a desired vector preferably an expression vector such as pKK223-3
  • the promoter sequence and the terminator sequence to be in the shuffled DNA are not limited to only one each, but a plurality of promoter sequences and terminator sequences may be therein.
  • the polynucleotide blocks positioned at the both ends of the shuffled DNA may be designed to have suitable restriction enzyme recognizing sites.
  • the DNA may be ligated to a suitable vector, using the defined re- striction enzymes.
  • the vector library thus produced in the manner mentioned above is introduced into a suitable host, in which the genetic information is expressed.
  • the intended gene- tic product with favorable properties and also the gene coding for it can be obtained.
  • Any and every ordinary host can be used herein.
  • Preferred examples of the host include cells of E. coli , bacillus bacteria, yeasts, and lactic acid bacteria. If desired, in-vitro transcription systems and translation systems are also employable herein. In those cases, the genetic information can be expressed even when the gene is not ligated to a vector.
  • the "genetic information” as referred to herein indicates the information on a gene which is carried by a DNA and which is translated into a protein or is transcribed into RNA in a suitable living body by the DNA for itself or after having been ligated to any other DNA or RNA.
  • the genetic information that is expected to be expressed according to the method of the present invention is not specifically defined, but includes, for example, those on various genetic products, such as enzymes, antibodies, hormones receptor proteins and ribozymes, and those on various control functions of, for example, operators, promoters and attenua- tors.
  • a nucleic acid pool was produced in accordance with the process mentioned below, based on the wild-type alkali protease
  • Step a) Preparation of Oligonucleotide Blocks for Primer (1-1) Synthesis of Oligonucleotide Blocks:
  • oligonucleotides containing a part of the base sequence are parts of the base sequence of API21 (Japanese Patent Application Laid-Open No. 5-91876) (including complementary strands) , or oligonucleotides containing a part of the base sequence.
  • the sequence of oligo 4a is to follow glutamine of Sequence Number 1 and, and this contains a termination codon of the gene.
  • These oligonucleotides were so designed that they might be the best when the oligo A was overhung on the 3 '-terminal of the amplified DNA in the experiment to follow hereinunder, using a Tag polymerase.
  • oligonucleotides were synthesized in a DM trityl-on condition (that is, while the 5' -hydroxyl group was protected with dimethoxytrityl group) , and purified through an OPC column.
  • the reagents used herein were obtained from Perkin Elmer Co.
  • HCC hexaammine cobalt chloride
  • BSA bovine serum albumin
  • Oligo 2r, oligo 3r, oligo 4r, oligo lb, oligo 2b, oligo 3b and oligo 4b were processed in the same manner as above. These four polynucleotides thus formed are referred to as oligo lr ' , oligo 2r', oligo 3r', oligo 4r', oligo lb', oligo 2b', oligo 3b' and oligo 4b' . (1-3) Phosphorylation:
  • Oligo 2a, oligo 3a and oligo 4a were processed in the same manner as above. These polynucleotides thus formed are referred to as oligo la', oligo 2a', oligo 3a' and oligo 4a'. (1-4) Ligation of Oligonucleotide Blocks:
  • oligo la' 100 pmols of oligo lb', 100 pmols of oligo 2b', 100 pmols of oligo 3b', 100 pmols of oligo 4b', which had been obtained in the above, as well as 1 nmol of ATP and 50 units of T4 RNA ligase were added to the same standard solution as that mentioned above to make 10 ⁇ l in total, and these were reacted at 25°C for 4 hours.
  • oligo 2a' with oligo lb', oligo 2b', oligo 3b' and oligo 4b' were also reacted in the same manner as above.
  • oligo 1M A mixture of the four polynucleotides thus formed as a result of this reaction, oligo la' ligated to oligo lb', oligo 2b', oligo 3b' and oligo 4b', is referred to as oligo 1M; a mixture of the four polynucleotides, oligo 2a' ligated to oligo lb', oligo 2b', oligo 3b' and oligo 4b', is referred to as oligo 2M; a mixture of the four polynucleotides, oligo 3a' ligated to oligo lb', oligo 2b', oligo 3b' and oligo 4b', is referred to as oligo 3M; and a mixture of the four polynucleotides, oligo 4a' ligated to oligo lb', oligo 2b', oligo 3b' and oligo
  • Steps b) to d) Formation of Gene Blocks
  • plasmid pSDT812 Japanese Patent Application Laid-Open No. 1-141596
  • the gene of the wild- type alkali protease as cloned from Bacillus NKS-21 was subjected to PCR with primers, oligo 1M and oligo 2r'.
  • the gene fragment as amplified through this reaction was treated with a ribonuclease, and then heated at 80_C for 5 minutes, whereby the polynucleotide (s) positioned at the 5 '-terminal of the ribonucleotide existing in the both strands or one strand was/were removed.
  • prepared was a gene block with cohesive end(s) .
  • This gene block is referred to as block 1M.
  • Block 1M, block 2M, block 3M, block B and block F of the same amount were blended and ligated together, using Pfu DNA ligase.
  • the reaction mixture was subjected to agarose gel electrophoresis, through which was collected the DNA fragment of about 1.5 kbp.
  • the thus-collected DNA of about 1.5 kbp was digested with restriction enzymes, EcoRI and BamHI, then mixed with a plasmid, pHY300PLK (manufactured by Yakulto Honsha Co.), which had been digested with restriction enzymes, EcoRI and BamHI and processed with an alkali phosphatase, and thus ligated together, using a ligation kit (manufactured by Takara Shuzo Co.).
  • a ligation kit manufactured by Takara Shuzo Co.
  • nucleic acid pool is obtained that covers all combinations of clones each containing the same or different three of these blocks.
  • the DNAs as produced in Example 3 were mixed. Using the resulting DNA mixture, cells of Bacillus subtili ⁇ UOT0999 were transformed. Tetracycline-resistant transformants were selec- ted. 300 transformants were replicated on a skim milk-containing medium plate, on which were found clear zones around the colonies of 12 transformants. Accordingly, it is under- stood that the enzyme which the shuffled gene codes for can be selected depending on its activity. The base sequences of these 12 clones that formed the clear zones were analyzed, from which it was found that these were sequenced in the same order of blocks as in the wild-type enzyme.
  • RNAs From 10 clones (one clone forms halo, while 9 clones do not) as selected from the transformants that had obtained in Example 3, and also from the host, Bacillus subtilis U0T0999, full-length RNAs were prepared. These were proces ⁇ ed with a ribonuclease-free deoxyribonuclease, in order to remove the influence of the plasmids on the hybridization to be effected later on. Next, using oligo lr as the probe, these were subj- ected to Northern hybridization. As a result, all lanes corresponding to the RNA of the transformants gave detectable bands, but no band was detected on the lanes corresponding to the RNA of the host.
  • a double- stranded DNA molecule with any desired cohesive end or ends.
  • various DNAs with various base sequences which are substantially apart from the naturally-existing base seguence spaces, and also a DNA pool of a mixture of such DNAs, through simple processes. Therefore, it is possible to obtain excellent genetic products, such as proteins and enzymes, which could not be obtained in conventional methods and which were not examined by organisms in the past.
  • nucleic acid pool it is possible to obtain a mixture of nucleic acids while optionally shuffling the constitutive blocks at random in the intermediate parts but fixing the terminal sequences to be predetermined, desired ones, and it is also possible to shuffle the constitutive blocks without changing the amino acid sequence which each block codes for. Therefore, as compared with a me- thod of producing a completely-randomized nucleic acid pool, there is a high possibility that useful genetic products can be produced according to the method of the present invention.
  • Length of Sequence 16 Type of Sequence: Nucleic Acid Number of Strand: Single-stranded Topology : Linear
  • Fig. 1 is a graphical view showing one embodiment of trie method of the present invention for shuffling a DNA.

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Abstract

L'invention porte sur un procédé de mutation d'ADN sensiblement différent des procédés usuels applicables aux ADN naturels et d'obtention de produits génétiques utiles à l'aide des ADN ainsi mutés. L'invention porte également sur un ADN à extrémité cohésive comprenant: (a) un ADN double brin présentant la même séquence qu'une partie de gène, et (b) un ADN monobrin présentant soit une séquence de base existant dans le même gène mais dans un site non contigu à la partie correspondant audit ADN double brin, soit une séquence de base que ne présente pas ledit gène, l'ADN monobrin étant fixé à l'une des extrémités de l'ADN double brin pour former une extrémité cohésive. Elle porte en outre sur son procédé d'obtention, sur un procédé de réarrangement d'un ADN l'utilisant, sur un ADN et un ensemble d'ADN obtenu à l'aide du procédé de réarrangement, sur un procédé d'obtention de l'ensemble d'ADN, et sur un produit génétique s'obtenant par expression des informations génétiques présentes dans l'ensemble d'ADN.
PCT/DK1997/000317 1996-08-07 1997-07-23 Adn double brin a extremite(s) cohesive(s) et procede de rearrangement d'adn y recourant WO1998005765A1 (fr)

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US6248541B1 (en) 2000-04-21 2001-06-19 Genencor International, Inc. Screening under nutrient limited conditions
WO2001081568A1 (fr) * 2000-04-21 2001-11-01 Genencor International, Inc. Reconstruction d'acides nucleiques n'utilisant pas la pcr
WO2002018629A1 (fr) * 2000-08-29 2002-03-07 Macquarie University Rearrangement d'un gene oligonucleotidique degenere
EP1192280A1 (fr) * 1999-06-14 2002-04-03 Diversa Corporation Reassemblage synthetique par ligature a evolution dirigee
US6534292B1 (en) 2000-05-08 2003-03-18 Genencor International, Inc. Methods for forming recombined nucleic acids
US6582914B1 (en) 2000-10-26 2003-06-24 Genencor International, Inc. Method for generating a library of oligonucleotides comprising a controlled distribution of mutations
US6808904B2 (en) 1994-06-16 2004-10-26 Syngenta Participations Ag Herbicide-tolerant protox genes produced by DNA shuffling
EP1482433A2 (fr) * 2001-08-10 2004-12-01 Xencor Automatisation de conception de protéines pour la préparation de bibliothèques de protéines
US6994963B1 (en) 2000-07-10 2006-02-07 Ambion, Inc. Methods for recombinatorial nucleic acid synthesis
WO2006047669A2 (fr) * 2004-10-27 2006-05-04 Monsanto Technology Llc Methode de modification genique non aleatoire
US7315786B2 (en) 1998-10-16 2008-01-01 Xencor Protein design automation for protein libraries
US7379822B2 (en) 2000-02-10 2008-05-27 Xencor Protein design automation for protein libraries
WO2013032850A3 (fr) * 2011-08-26 2013-04-25 Gen9, Inc. Compositions et procédés pour un assemblage haute-fidélité d'acides nucléiques
AU2009212959B2 (en) * 1999-06-14 2013-08-15 Bp Corporation North America Inc. Synthetic ligation reassembly in directed evolution
US9217144B2 (en) 2010-01-07 2015-12-22 Gen9, Inc. Assembly of high fidelity polynucleotides
US9216414B2 (en) 2009-11-25 2015-12-22 Gen9, Inc. Microfluidic devices and methods for gene synthesis
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US10202608B2 (en) 2006-08-31 2019-02-12 Gen9, Inc. Iterative nucleic acid assembly using activation of vector-encoded traits
US10207240B2 (en) 2009-11-03 2019-02-19 Gen9, Inc. Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
US10308931B2 (en) 2012-03-21 2019-06-04 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
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CN111019938A (zh) * 2019-12-06 2020-04-17 天津大学 一种带有粘性末端的长链dna及制备方法
US11072789B2 (en) 2012-06-25 2021-07-27 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
US11084014B2 (en) 2010-11-12 2021-08-10 Gen9, Inc. Methods and devices for nucleic acids synthesis

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US7315786B2 (en) 1998-10-16 2008-01-01 Xencor Protein design automation for protein libraries
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AU2009212959B2 (en) * 1999-06-14 2013-08-15 Bp Corporation North America Inc. Synthetic ligation reassembly in directed evolution
EP2865754A1 (fr) * 1999-06-14 2015-04-29 BP Corporation North America Inc. Réassemblage par ligature synthétique dans une évolution dirigée
USRE45349E1 (en) 1999-06-14 2015-01-20 Bp Corporation North America Inc. Synthetic ligation reassembly in directed evolution
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US7379822B2 (en) 2000-02-10 2008-05-27 Xencor Protein design automation for protein libraries
US6248541B1 (en) 2000-04-21 2001-06-19 Genencor International, Inc. Screening under nutrient limited conditions
WO2001081568A1 (fr) * 2000-04-21 2001-11-01 Genencor International, Inc. Reconstruction d'acides nucleiques n'utilisant pas la pcr
US6534292B1 (en) 2000-05-08 2003-03-18 Genencor International, Inc. Methods for forming recombined nucleic acids
US7037726B2 (en) 2000-05-08 2006-05-02 Genencor International, Inc. Methods for forming recombined nucleic acids
US6994963B1 (en) 2000-07-10 2006-02-07 Ambion, Inc. Methods for recombinatorial nucleic acid synthesis
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US6582914B1 (en) 2000-10-26 2003-06-24 Genencor International, Inc. Method for generating a library of oligonucleotides comprising a controlled distribution of mutations
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