Nucleic acid synthesis methods
First Claim
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1. A method comprising the steps:
- a) providing a sequence of a defined nucleic acid molecule to be synthesized,b) providing an oligonucleotide produced by the following steps;
ba) providing a partially double-stranded first oligonucleotide with a 5′
-overhang comprising a recognition site for a first type IIS restriction enzyme which cuts outside of its recognition site, wherein the first oligonucleotide comprises a modification which allows coupling to a solid matrix, wherein the 5′
-overhang has a length of 3 nucleotides,bb) providing a partially double-stranded second oligonucleotide with a 5′
-overhang comprises a recognition site for a second type IIS restriction enzyme which cuts outside of its recognition site, the recognition site for the first and second type IIS restriction enzymes being different, wherein the 5′
-overhang has a length of 3 nucleotides,bc) ligating the first and second oligonucleotides from steps ba) and bb) in the orientation defined by the blocking of the ends not to be ligated,bd) removing unconsumed reactants as well as enzymes,be) cleaving the ligation product from step bc) with the second type IIS restriction enzyme which cuts outside of its recognition site, wherein the cleavage occurs in the nucleic acid sequence of the second oligonucleotide from step bb), creating a first elongated product, wherein the first elongated product comprises the first oligonucleotide from step ba) which has been elongated, bf) separating the reaction mixture from the first elongated product,c) providing a further oligonucleotide produced by the following steps;
ca) providing a partially double-stranded third oligonucleotide with a 5′
-overhang, comprising a recognition site for a third type IIS restriction enzyme which cuts outside of its recognition site, wherein the third oligonucleotide comprises a modification which allows coupling to a solid matrix, wherein the 5′
-overhang has a length of 3 nucleotides,cb) providing a partially double-stranded fourth oligonucleotide with a 5′
-overhang comprising a recognition site for a fourth type IIS restriction enzyme which cuts outside of its recognition site, the recognition sites of the third and fourth type IIS restriction enzymes being different, wherein the 5′
-overhang has a length of 3 nucleotides,cc) ligating the third and fourth oligonucleotides from steps ca) and cb) in the orientation defined by the blocking of the ends not to be ligated,cd) removing unconsumed reactants as well as enzymes,ce) cleaving the ligation product from step cc) with the fourth type IIS restriction enzyme, which cuts outside of its recognition site, whereby the cleavage occurs in the fourth oligonucleotide in step cb), creating a second elongated product, wherein the second elongated product comprises the third oligonucleotide from step ca) which has been elongated,cf) separating the reaction mixture from the second elongated product,d) ligating the first and second elongated products from steps b) and c) in the orientation defined by the blocking of the ends not to be ligated,e) removing unconsumed reactants as well as enzymes,f) cleavage of the ligation product from step d) with a type IIS restriction enzyme which cuts outside of its recognition site, whereby the cleavage occurs in the first or second elongated product from steps b) or c), thus creating a third elongated product,g) separating the third elongated product from the reaction mixture, characterized in that the second oligonucleotide from step bb) has the recognition site for a type IIS restriction enzyme which generates an overhang three nucleotides in length as long as steps bb) to be) are repeated and the second oligonucleotide from step bb) has the recognition site for a type IIS restriction enzyme which generates an overhang other than an overhang three nucleotides in length, in the last cycle of the steps bb) to be) and/or the fourth oligonucleotide from step cb) has the recognition site for a type IIS restriction enzyme which generates an overhang three nucleotides in length, as long as steps cb) to ce) are repeated and the fourth oligonucleotide from step cb) has the recognition site of a type IIS restriction enzyme, which produces an overhang other than an overhang three nucleotides in length, in the last cycle of the steps cb) to ce) andh) isolating the nucleic acid molecule or part thereof having the sequence provided in step a).
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Abstract
The present invention relates to a single-stranded nucleic acid molecule for use in a method for the production of a nucleic acid, whereby the nucleic acid molecule comprises a part A and a part B, whereby part A comprises a sequence, which corresponds at least to a partial sequence of the recognition site of a type IIS restriction enzyme, and part B comprises an arbitrary but defined sequence of nucleotides. By using such nucleic acid molecules it is possible to assemble different fragments in a sequence-independent manner and thus conduct the synthesis of a nucleic acid with recourse to standardized elements.
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Citations
21 Claims
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1. A method comprising the steps:
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a) providing a sequence of a defined nucleic acid molecule to be synthesized, b) providing an oligonucleotide produced by the following steps; ba) providing a partially double-stranded first oligonucleotide with a 5′
-overhang comprising a recognition site for a first type IIS restriction enzyme which cuts outside of its recognition site, wherein the first oligonucleotide comprises a modification which allows coupling to a solid matrix, wherein the 5′
-overhang has a length of 3 nucleotides,bb) providing a partially double-stranded second oligonucleotide with a 5′
-overhang comprises a recognition site for a second type IIS restriction enzyme which cuts outside of its recognition site, the recognition site for the first and second type IIS restriction enzymes being different, wherein the 5′
-overhang has a length of 3 nucleotides,bc) ligating the first and second oligonucleotides from steps ba) and bb) in the orientation defined by the blocking of the ends not to be ligated, bd) removing unconsumed reactants as well as enzymes, be) cleaving the ligation product from step bc) with the second type IIS restriction enzyme which cuts outside of its recognition site, wherein the cleavage occurs in the nucleic acid sequence of the second oligonucleotide from step bb), creating a first elongated product, wherein the first elongated product comprises the first oligonucleotide from step ba) which has been elongated, bf) separating the reaction mixture from the first elongated product, c) providing a further oligonucleotide produced by the following steps; ca) providing a partially double-stranded third oligonucleotide with a 5′
-overhang, comprising a recognition site for a third type IIS restriction enzyme which cuts outside of its recognition site, wherein the third oligonucleotide comprises a modification which allows coupling to a solid matrix, wherein the 5′
-overhang has a length of 3 nucleotides,cb) providing a partially double-stranded fourth oligonucleotide with a 5′
-overhang comprising a recognition site for a fourth type IIS restriction enzyme which cuts outside of its recognition site, the recognition sites of the third and fourth type IIS restriction enzymes being different, wherein the 5′
-overhang has a length of 3 nucleotides,cc) ligating the third and fourth oligonucleotides from steps ca) and cb) in the orientation defined by the blocking of the ends not to be ligated, cd) removing unconsumed reactants as well as enzymes, ce) cleaving the ligation product from step cc) with the fourth type IIS restriction enzyme, which cuts outside of its recognition site, whereby the cleavage occurs in the fourth oligonucleotide in step cb), creating a second elongated product, wherein the second elongated product comprises the third oligonucleotide from step ca) which has been elongated, cf) separating the reaction mixture from the second elongated product, d) ligating the first and second elongated products from steps b) and c) in the orientation defined by the blocking of the ends not to be ligated, e) removing unconsumed reactants as well as enzymes, f) cleavage of the ligation product from step d) with a type IIS restriction enzyme which cuts outside of its recognition site, whereby the cleavage occurs in the first or second elongated product from steps b) or c), thus creating a third elongated product, g) separating the third elongated product from the reaction mixture, characterized in that the second oligonucleotide from step bb) has the recognition site for a type IIS restriction enzyme which generates an overhang three nucleotides in length as long as steps bb) to be) are repeated and the second oligonucleotide from step bb) has the recognition site for a type IIS restriction enzyme which generates an overhang other than an overhang three nucleotides in length, in the last cycle of the steps bb) to be) and/or the fourth oligonucleotide from step cb) has the recognition site for a type IIS restriction enzyme which generates an overhang three nucleotides in length, as long as steps cb) to ce) are repeated and the fourth oligonucleotide from step cb) has the recognition site of a type IIS restriction enzyme, which produces an overhang other than an overhang three nucleotides in length, in the last cycle of the steps cb) to ce) and h) isolating the nucleic acid molecule or part thereof having the sequence provided in step a). - View Dependent Claims (2)
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3. A method comprising the steps:
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a) providing a sequence of a defined nucleic acid molecule to be synthesized, b) providing an oligonucleotide produced by the following steps; ba) providing a partially double-stranded first oligonucleotide comprising a 5′
overhang and a recognition site for a first type IIS restriction enzyme which cuts outside of its recognition site, wherein the 5′
overhang has a length of 3 nucleotides,bb) providing a partially double-stranded second oligonucleotide comprising a 5′
overhang and a recognition site for a second type IIS restriction enzyme which cuts outside of its recognition site, the recognition sites of the first and second type IIS restriction enzymes being different, wherein the second oligonucleotide comprises a modification allowing coupling to a solid matrix, wherein the 5′
overhang has a length of 3 nucleotides,bc) ligating the first and second oligonucleotides from steps ba) and bb) in the orientation defined by the blocking of the ends not to be ligated, bd) cleaving the ligation product from step be) with the second type IIS restriction enzyme which cuts outside of its recognition site, wherein the cleavage occurs in the nucleic acid sequence of the second oligonucleotide from step bb), creating a first elongated product, wherein the first elongated product comprises the first oligonucleotide from step ba) which has been elongated, bf) separating from the reaction mixture the first elongated product, c) providing a further oligonucleotide produced by the following steps; ca) providing a third oligonucleotide comprising a 5′
overhang and a recognition site for a third type IIS restriction enzyme, wherein the 5′
overhang has a length of 3 nucleotides,cb) providing a partially double-stranded fourth oligonucleotide comprising a 5′
overhang and a recognition site for a fourth type IIS restriction enzyme which cuts outside of its recognition site, the recognition sites of the third and fourth type IIS restriction enzymes being different, wherein the fourth oligonucleotide comprises a modification which allows coupling to a solid matrix, wherein the 5′
overhang has a length of 3 nucleotides,cc) ligating the third and fourth oligonucleotides from steps ca) and cb) in the orientation defined by the blocking of the ends not to be ligated, ce) cleaving the ligation product from step cc) with a fourth type IIS restriction enzyme which cuts outside of its recognition site, whereby the cleavage occurs in the fourth oligonucleotide from step cb), creating a second elongated product, wherein the second elongated product comprises the third oligonucleotide from step ca) which has been elongated, cf) separating from the reaction mixture the second elongated product from step ce), d) ligating the first and second elongated products from steps b) and c) in the orientation defined by the blocking of the ends not to be ligated, e) removing and/or inactivating unconsumed reactants as well as enzymes, f) cleaving the ligation product from step d) with a type IIS restriction enzyme which cuts outside of its recognition site, whereby the cleavage occurs in the first or second elongated product from steps b) or c), creating a third elongated product, g) separating the third elongated product from the reaction mixture, and h) isolating the nucleic acid molecule or part thereof having the sequence provided in step a). - View Dependent Claims (4, 5, 6)
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7. A method comprising the steps:
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a) providing a sequence of a defined nucleic acid molecule to be synthesized, b) providing an oligonucleotide produced by the following steps; ba) providing a first oligonucleotide comprising a 5′
overhang and a recognition site for a first type IIS restriction enzyme which cuts outside of its recognition site, wherein the first oligonucleotide comprises a modification which allows coupling to a solid matrix, and coupling the first oligonucleotide to the solid matrix, wherein the 5′
overhang has a length of 3 nucleotides,bb) providing a partially double-stranded second oligonucleotide comprising a 5′
overhang and a recognition site for a second type IIS restriction enzyme which cuts outside of its recognition site, the recognition site for the first and second type II restriction enzymes being different, wherein the 5′
overhang has a length of 3 nucleotides,bc) ligating the first and second oligonucleotides from steps ba) and bb) in the orientation defined by the blocking of the ends not to be ligated, bd) removing unconsumed reactants as well as enzymes, be) cleaving the ligation product from step be) with the second type IIS restriction enzyme which cuts outside of its recognition site, wherein the cleavage occurs in the nucleic acid sequence of the second oligonucleotide from step bb), creating a first elongated product, wherein the first elongated product comprises the first oligonucleotide from step ba) which has been elongated, bf) separating the reaction mixture from the first elongated product, c) providing a further oligonucleotide produced by the steps; ca) providing a third oligonucleotide comprising a 5′
overhang and a recognition site for a third type IIS restriction enzyme which cuts outside of its recognition site, wherein the third oligonucleotide comprises a modification which allows coupling to a solid matrix, coupling of the third oligonucleotide to the solid matrix, wherein the 5′
overhang has a length of 3 nucleotides,cb) providing a partially double-stranded fourth oligonucleotide comprising a 5′
overhang and a recognition site for a fourth type IIS restriction enzyme which cuts outside of its recognition site, the recognition site of the third and fourth type IIS restriction enzymes being different, wherein the 5′
overhang has a length of 3 nucleotides,cc) ligation of the third and fourth oligonucleotides from steps ca) and cb) in the orientation defined by the blocking of the ends not to be ligated, cd) removing unconsumed reactants as well as enzymes, ce) cleavage of the ligation product from step cc) with the fourth type IIS restriction enzyme which cuts outside of its recognition site, whereby the cleavage occurs in the fourth oligonucleotide in step cb), creating a second elongated product, wherein the second elongated product comprises the third oligonucleotide from step ca) which has been elongated, cf) separating the second elongated product from the reaction mixture, d) ligating the first and second elongated products from steps b) and c) in the orientation defined by the blocking of the ends not to be ligated, e) removing unconsumed reactants as well as enzymes, f) cleavage of the ligation product from step d) with a type IIS restriction enzyme, which cuts outside of its recognition site, whereby the cleavage occurs in the first or second elongated product from steps b) or c), creating a third elongation product, g) separating the third elongation product from the reaction mixture, wherein the last repetition of steps bb) to bf) the first oligonucleotide added in step bb) carries a modification, which allows coupling to a solid matrix, and h) isolating the nucleic acid molecule or part thereof having the sequence provided in step a). - View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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Specification