Nucleic Acid Linkers and Use Thereof in Gene Synthesis

• US 20080044862A1
• Filed: 11/22/2002
• Published: 02/21/2008
• Est. Priority Date: 11/22/2001
• Status: Active Grant

• Current Assignee: Morphosys Ag
• Original Assignee: Morphosys Ag
• Inventors: O'Connell, Timothy, Schatz, Octavian
• Granted Patent: US 9,957,502 B2
• Time in Patent Office: Days
• US Class Current: 435/85
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/66 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
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

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.

Claims

1-99. -99. (canceled)

100. A method for the production of a nucleic acid molecule comprising the steps

a) providing an oligonucleotide, generated by the following steps:

aa) providing a partially double-stranded oligonucleotide with a 5′

-overhang, containing a recognition site for a type IIS restriction enzyme cleaving outside of its recognition site, and carrying a modification, which allows coupling to a solid matrix, whereby the 5′

-overhang has a length of 3 nucleotides,

ab) addition of a further, at least partially double-stranded oligonucleotide with a 5′

-overhang and a different recognition site for a type IIS restriction enzyme, which cleaves outside of its recognition site, than in step aa), whereby the 5′

-overhang has a length of 3 nucleotides,

ac) ligation of the oligonucleotides from step aa) and ab) in the orientation defined by the blocking of the ends not to be ligated,

ad) removing unused reactants as well as enzymes,

ae) cleavage of the ligation product from step ac) with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the nucleic acid sequence of the oligonucleotide from step ab),

af) separation of the reaction mixture from the elongated oligonucleotide obtained in step ae), which had been provided in step aa),

ag) optionally repeating steps ab) to af) at least one time,

b) providing a further oligonucleotide, generated by the steps;

ba) providing a partially double-stranded oligonucleotide with a 5′

-overhang, containing a recognition site for a type IIS restriction enzyme, which cuts outside of its recognition site, and carrying a modification, which allows coupling to a solid matrix, whereby the 5′

-overhang has a length of 3 nucleotides,

bb) addition of a further, at least partially double-stranded oligonucleotide with a 5′

-overhang and with a different recognition site for a type IIS restriction enzyme, which cleaves outside of its recognition site, than in step ba), whereby the 5′

-overhang has a length of 3 nucleotides,

bc) ligation of the oligonucleotides from step ba) and bb) in the orientation defined by the blocking of the ends not to be ligated,

bd) removing unused reactants as well as enzymes,

be) cleavage of the ligation product from step bc) with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the nucleic acid sequence of the oligonucleotide from step bb),

bf) separation of the thus elongated oligonucleotide from the reaction mixture,

bg) optionally repeating steps bb) to be) at least one time, whereby subsequent to the last ligation in step bc) and removal of unused reactants as well as enzymes, the ligation product is cleaved with a type IIS restriction enzyme, whereby the cleavage occurs in the oligonucleotide from step ba),

c) ligation of the oligonucleotides from step a) and b) in the orientation defined by the blocking of the ends not to be ligated,

d) removal of unused reactants as well as enzymes,

e) cleavage of the ligation product from step c) with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the oligonucleotide from step a) or b),

f) separating the thus elongated nucleic acid molecule from the reaction mixture, characterized in that the oligonucleotide of step ab) contains the recognition site for a type IIS restriction enzyme, which generates an overhang three nucleotides in length as long as steps

ab) to ae) are repeated and the oligonucleotide of step ab) possesses the recognition site of a type IIS restriction enzyme, which produces an overhang other than three nucleotides in length, in the last cycle of the steps ab) to ae) and/or

the oligonucleotide from step bb) contains 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 oligonucleotide of step bb) possesses the recognition site of a type IIS restriction enzyme, which produces an overhang other than three nucleotides in length, in the last cycle of the steps bb) to be).

101. The method according to claim 100, characterized in that the oligonucleotide of step ab) and/or of step bb) is an oligonucleotide according to one of the claims 4 to 14, as long as the cycle of steps ab) to ae) and/or bb) to be) is not the last cycle.

102. The method according to claim 100 or 101, characterized in that the oligonucleotide of step ab) and/or of step bb) in the last cycle of steps ab) to ae) and/or bb) to be) is an oligonucleotide according to one of the claims 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

103. A method for the production of a group of nucleic acid molecules comprising the steps

a) providing an oligonucleotide, generated by the following steps:

aa) Providing an oligonucleotide, containing a recognition site for a type IIS restriction enzyme cutting outside of its recognition site, and carrying a modification, which allows coupling to a solid matrix,

coupling the oligonucleotides to the solid matrix,

ab) addition of a further, at least partially double-stranded oligonucleotide with a different recognition site for a type IIS restriction enzyme, cutting outside of its recognition site, than in step aa),

ac) ligation of the oligonucleotides from step aa) and ab) in the orientation defined by the blocking of the ends not to be ligated,

ad) removing unused reactants as well as enzymes,

ae) cleavage of the ligation product from step ac) with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the nucleic acid sequence of the oligonucleotide from step ab),

af) separating of the reaction mixture from the elongated oligonucleotide obtained in step ae), first provided in step aa),

ag) optionally repeating steps ab) to af) at least one time,

b) providing a further oligonucleotide, generated by the steps;

ba) providing a partially double-stranded oligonucleotide, containing a recognition site for a type IIS restriction enzyme, which cuts outside of its recognition site, and carrying a modification, which allows coupling to a solid matrix, with one end to a solid matrix,

coupling of the oligonucleotide to the solid matrix,

bb) addition of a further, at least partially double-stranded oligonucleotide with a different recognition site for a type IIS restriction enzyme than in step ba), which cleaves outside of its recognition site,

bc) ligation of the oligonucleotides from step ba) and bb) in the orientation defined by the blocking of the ends not to be ligated,

bd) removing unused reactants as well as enzymes,

be) cleavage of the ligation product from step bc) with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the nucleic acid sequence of the oligonucleotide from step bb),

bf) separating the thus elongated oligonucleotide from the reaction mixture,

bg) optionally repeating steps bb) to bf) at least one time, whereby subsequent to the last ligation in step bc) and removing unused reactants as well as enzymes the ligation product is cleaved with a type IIS restriction enzyme, whereby the cleavage occurs in the oligonucleotide from step ba),

c) ligation of the oligonucleotides from step a) and b) in the orientation defined by the blocking of the ends not to be ligated,

d) removing unused reactants as well as enzymes,

e) cleavage of the ligation product from step c) with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the oligonucleotide from step a) or b),

f) separating the thus elongated nucleic acid molecules from the reaction mixture,

characterized in that in the last repetition of steps ab) to af) the oligonucleotide added in step ab) carries a modification, which allows coupling to a solid matrix and

after the last repetition of steps ab) to af), as step ah), the ligation product from step ac) is cut with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the nucleic acid sequence of the oligonucleotide from step aa), and the cleavage product of the oligonucleotide coupled to the solid matrix is released,

and the released cleavage product is divided in at least two reactions.

104. The method according to claim 103, characterized in that the cleavage product released in step ah) is coupled to a solid matrix via the modification, and that the cleavage products that do not contain the modification, are removed from the reaction.

105. The method according to claim 104, characterized in that, as step ai), in each of the reactions an oligonucleotide is added to the cleavage product coupled to the solid matrix from step ah), whereby this oligonucleotide contains a recognition site for a type IIS restriction enzyme, which is different from the recognition site of a type IIS restriction enzyme of the cleavage products from step ah), and carries a modification, which allows coupling to a solid matrix, and, as step ak), the cleavage products from step ah) are ligated with the oligonucleotide.

106. The method according to claim 105, characterized in that the ligation product from step ak) is cut with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the nucleic acid sequence of the cleavage product from step ah).

107. The method according to claim 105 or 106, characterized in that the oligonucleotide added in step ai) has another sequence in each reaction.

108. The method according to one of the claims 105, characterized in that the oligonucleotides added to the reactions have identical single-stranded overhangs.

109. The method according to one of the claims 104, 105, 106, 107 or 108, characterized in that the oligonucleotides differ in a region that is different from the single-stranded region, preferably in a region comprising a sequence of nucleotides following the single-stranded region of the oligonucleotide.

110. The method according to claim 109, characterized in that the sequence of nucleotides has a length of 1 to 10 nucleotides, preferably 3 to 6 nucleotides.

111. The method according to any one of claims 103, 104, 105, 106, 107, 108, 109 or 110, characterized in that, in a step al), the oligonucleotide added in step ai) is cleaved.

112. The method according to claim 111, characterized in that the steps ab) to ak) and/or al) are repeated at least once.

113. The method according to claim 112, characterized in that, after the last repetition, the oligonucleotides coupled to the solid phase are ligated, as step am), with a further oligonucleotide according to ab), whereby these oligonucleotides in the different reactions differ from each other in the sequence of the single-stranded overhang and optionally in the sequence of the directly abutting double-stranded region.

114-131. -131. (canceled)

132. A method for the production of a nucleic acid molecule comprising the steps of:

a) Providing an oligonucleotide, generated by the following steps;

aa) Providing an oligonucleotide, containing a recognition site for a type IIS restriction enzyme cutting outside of its recognition site,

ab) addition of a further, at least partially double-stranded oligonucleotide with a different recognition site for a type IIS restriction enzyme, cutting outside of its recognition site, than in step aa), and which carries a modification allowing coupling to a solid matrix,

ac) ligation of the oligonucleotides from step aa) and ab) in the orientation defined by the blocking of the ends not to be ligated,

ad) optionally removing and/or inactivating unused reactants as well as enzymes,

ae) cleavage of the ligation product from step ac) with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the nucleic acid sequence of the oligonucleotide from step ab),

af) separating the cleavage product from step ae) that does not carry a modification from the reaction mixture,

ag) optionally repeating steps ab) to af) at least one time,

b) providing a further oligonucleotide, generated by the steps;

ba) Providing an oligonucleotide, containing a recognition site for a type IIS restriction enzyme cutting outside of its recognition site,

coupling the oligonucleotide to the solid matrix,

bb) addition of a further, at least partially double-stranded oligonucleotide with a different recognition site for a type IIS restriction enzyme, cutting outside of its recognition site, than in step ba), and carrying a modification, which allows coupling to a solid matrix,

bc) ligation of the oligonucleotides from step ba) and bb) in the orientation defined by the blocking of the ends not to be ligated,

bd) optionally removing and/or inactivating unused reactants as well as enzymes,

be) cleavage of the ligation product from step bc) with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the nucleic acid sequence of the oligonucleotide from step bb),

bf) separating the cleavage product from step be) that does not carry a modification from the reaction mixture,

bg) optionally repeating steps bb) to bf) at least one time, whereby subsequent to the last ligation in step bc) and removing unused reactants as well as enzymes the ligation product is cleaved with a type IIS restriction enzyme, whereby the cleavage occurs in the oligonucleotide from step ba),

c) ligation of the oligonucleotides from step a) and b) in the orientation defined by the blocking of the ends not to be ligated in solution,

d) removing and/or inactivating unused reactants as well as enzymes,

e) cleavage of the ligation product from step c) with a type IIS restriction enzyme, which cleaves outside of its recognition site, whereby the cleavage occurs in the oligonucleotide from step a) or b),

f) separating the thus elongated nucleic acid molecule from the reaction mixture,

133. The method according to claim 132, further comprising the step that the cleavage products carrying no modification are transferred into a new reaction.

134-144. -144. (canceled)