Method for the synthesis of DNA fragments
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Abstract
The invention relates to a method that can be carried out in parallel and automated for the production of any nucleic acid, comprising the following steps: a) coupling an oligonucleotide to a solid matrix b) adding an additional oligonucleotide c) ligating the oligonucleotides from steps a) and b) in one orientation d) removing excess reactants and enzymes from the reaction preparation e) cleaving the ligation product from step c) with a restriction enzyme that cleaves outside the recognition sequence, whereby cleavage occurs in the oligonucleotide from step a) or in the oligonucleotide from step b) f) separating the reaction mixture from the lengthened or shortened oligonucleotide from step a) that is obtained in step e) g) repeating steps b) to f) at least once h) successive sequence-independent linkage of the fragments obtained after performing steps a) to g) until the desired product is obtained.
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Citations
37 Claims
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21. A method for the production of a nucleic acid molecule comprising the steps
a) providing an oligonucleotide which is prepared by the following steps: -
aa) coupling one end of an oligonucleotide to a solid matrix wherein the coupling is effected by means of a modification and the oligonucleotide contains a recognition sequence for a type IIS restriction enzyme which cleaves outside its recognition sequence, ab) adding an additional oligonucleotide separate from the oligonucleotide of aa) which is at least partially double-stranded and contains a different recognition sequence than in step aa) for a type IIS restriction enzyme which cleaves outside its recognition sequence, whereby this oligonucleotide cannot bind to the matrix, ac) ligating the oligonucleotides from steps aa) and ab) in an orientation determined by a blockage of ends of the oligonucleotides from steps aa) and ab) that are not to be ligated, ad) removing oligonucleotides from steps aa), ab) and ac) that are not coupled or ligated, ae) cleaving the ligation product from step ac) with a type IIS restriction enzyme which cleaves outside its recognition sequence whereby the cleavage occurs in the nucleic acid sequence of the oligonucleotide from step ab) and resulting in an elongated oligonucleotide and a shorter oligonucleotide, af) separating the type IIS restriction enzyme and the shorter oligonucleotide from the elongated oligonucleotide obtained in step ae), ag) repeating steps ab) to af) at least once, b) providing an additional oligonucleotide which is prepared by the following steps;
ba) coupling one end of an oligonucleotide to a solid matrix wherein the coupling is effected by means of a modification and the oligonucleotide contains a recognition sequence for a type IIS restriction enzyme which cleaves outside its recognition sequence, bb) adding an additional oligonucleotide separate from the oligonucleotide of ba) which is at least partially double-stranded and contains a different recognition sequence than in step ba) for a type IIS restriction enzyme which cleaves outside its recognition sequence, whereby this oligonucleotide cannot bind to the matrix, bc) ligating the oligonucleotides from steps ba) and bb) in an orientation determined by a blockage of ends of the oligonucleotides from steps ba) and bb) that are not to be ligated, bd) removing oligonucleotides from steps ba) and bb) that are not coupled or ligated, be) cleaving the ligation product from step bc) with a type IIS restriction enzyme which cleaves outside its recognition sequence whereby the cleavage occurs in the oligonucleotide from step bb) and resulting in an elongated oligonucleotide and a shorter oligonucleotide, bf) separating the type IIS restriction enzyme and the shorter oligonucleotide from the elongated oligonucleotide obtained in step be), bg) repeating steps bb) to bf) at least once, wherein after the last ligation in step bc) and removing oligonucleotides that are not coupled or ligated to obtain a ligation product, the ligation product is cleaved with a type IIS restriction enzyme whereby the cleavage occurs in the oligonucleotide from step ba), c) ligating the oligonucleotides from steps a) and b) in an orientation determined by a blockage of ends of the oligonucleotides from steps a) and b) that are not to be ligated, d) removing oligonucleotides from steps a), b) and c) that are not coupled or ligated, e) cleaving the ligation product from step c) with a type IIS restriction enzyme which cleaves outside its recognition sequence whereby the cleavage occurs in the oligonucleotide from step a) or b) and resulting in an elongated oligonucleotide and a shorter oligonucleotide, f) separating and removing the type IIS restriction enzyme and shorter oligonucleotide from the elongated oligonucleotide obtained from step e), wherein the elongated oligonucleotide is retained. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)
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Specification