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Nucleic acid synthesis methods

  • US 9,957,502 B2
  • Filed: 11/22/2002
  • Issued: 05/01/2018
  • Est. Priority Date: 11/22/2001
  • Status: Active Grant
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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