System and method of modular cloning

Abstract

System for producing a nucleic acid construct of interest, said system comprising: a set of n entry DNAs numbered 1 to n, n being an integer of at least 2, each of said n entry DNAs comprising in this order: (i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof; (ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and (iii) a cleavage site of a further type Ms restriction endonuclease recognition site followed by the recognition site of said cleavage site; the cleavage sites of the type IIs restriction endonuclease recognition sites of item (iii) of entry DNAs 1 to n−1 are complementary to the cleavage sites of the type IIs restriction endonuclease recognition sites of item (i) of entry DNAs 2 to n, respectively; the cleavage site of the type Ms restriction endonuclease recognition site of item (iii) of entry DNA n is complementary to the cleavage site of the type IIs restriction endonuclease recognition site of item (i) of entry DNA 1 for allowing annealing of complementary single-stranded overhangs formed by restriction at recognition site (i) of entry DNA 1 and at recognition site (iii) of entry DNA n; said system further comprising a destination vector comprising in this order: (I) a type Ms restriction endonuclease recognition site followed by the cleavage site thereof; (II) a vector backbone preferably comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III); (III) a further cleavage site of a type Ms restriction endonuclease recognition site followed by the recognition site of said cleavage site, and (IV) optionally, an insert between the recognition sites of item (III) and item (i); said cleavage sites of items (I) and (III) being different and non-complementary, said recognition sites of items (I) and (III) being preferably recognitions sites of the same endonuclease.

1 . System for producing a nucleic acid construct of interest, said system comprising:
 a set of n entry DNAs numbered 1 to n, n being an integer of at least 2, preferably at least 3,   each of said n entry DNAs comprising in this order:   (i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;   (ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and   (iii) a cleavage site of a further type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site;   the cleavage sites of the type IIs restriction endonuclease recognition site(s) of item (iii) of entry DNA(s) 1 to n−1 is/are complementary to the cleavage site(s) of the type IIs restriction endonuclease recognition site(s) of item (i) of entry DNA(s) 2 to n, respectively;   the cleavage site of the type IIs restriction endonuclease recognition site of item (iii) of entry DNA n is complementary to the cleavage site of the type IIs restriction endonuclease recognition site of item (i) of entry DNA 1;   said system further comprising a destination vector comprising in this order:   (I) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;   (II) a vector backbone comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III);   (III) a further cleavage site of a type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site, and   (IV) optionally, an insert between the recognition sites of item (III) and item (I).   
     
     
         2 . The system according to  claim 1 , wherein a type IIs restriction endonuclease recognising the recognition site (I) of said destination vector can produce a single-stranded overhang from the cleavage site of item (I) that is complementary to the single-stranded overhang producible by the type IIs restriction endonuclease recognising the recognition site (i) of entry DNA numbered 1 for enabling annealing of said complementary single-stranded overhangs and ligation of said destination vector with the DNA segment of item (ii) from entry DNA numbered 1. 
     
     
         3 . The system according to  claim 1 , said system comprising a nucleic acid linker comprising in the following order:
 (a) a type IIs restriction endonuclease recognition site;   (b) a cleavage site of said recognition site of item (a);   (c) a cleavage site of a further type IIs restriction endonuclease recognition site of the following item (d);   (d) a type IIs restriction endonuclease recognition site defining the cleavage site of item (c) and being a recognition site of a type IIs restriction endonuclease different from that of item (a);   (e) a type IIs restriction endonuclease recognition site, preferably of the same endonuclease as the recognition site of item (d);   (f) a cleavage site of said recognition site of item (e);   (g) a cleavage site of a further type IIs restriction endonuclease recognition site of the following item (h);   (h) a type IIs restriction endonuclease recognition site defining the cleavage site of item (g), preferably of the same endonuclease as the recognition site of item (a);
 said linker being capable of linking a cleavage site of item (iii) of one of a entry DNA numbered 1 to n, preferably of number 1 to n−1, to a cleavage site of item (III) of said destination vector. 
   
     
     
         4 . The system according to  claim 3 , wherein the cleavage site of item (iii) of one of said entry DNAs is complementary to the cleavage site of item (b) of said linker, and
 the cleavage site of item (g) of said linker is complementary to the cleavage site of item (III) of said destination vector.   
     
     
         5 . The system according to  claim 1 , comprising from 1 to n multiple destination vectors numbered 1 to n, each of said 1 to n destination vectors having segments (I) to (III) as defined in  claim 1  and optionally a segment (IV) as defined in  claim 1 ,
 wherein the cleavage sites of item (III) of all n destination vectors are identical and all cleavage sites of item (I) of all n destination vectors are unique among the cleavage sites of item (I). 
 
     
     
         6 . The system according to  claim 3 , comprising a set of n nucleic acid linkers numbered 1 to n, each n-th linker comprising items (a) to (h) as defined in  claim 3 ,
 the cleavage site of item (iii) of each n-th entry DNA is complementary to the cleavage site of item (b) of the n-th linker;   the cleavage site of item (g) of each n-th linker being complementary to the cleavage site of item (III) of the n-th destination vector;   whereby each n-th linker being capable of linking a cleavage site of item (iii) of the n-th entry DNA to a cleavage site of item (III) of each n-th destination vector.   
     
     
         7 . The system according to  claim 1 , wherein each sequence portion of item (ii) of each entry DNA 1 to n comprises a further pair of two type IIs restriction endonuclease recognition sites oriented such that said further pair of recognition sites can be removed from said entry DNAs by treatment with type IIs restriction endonuclease(s) recognising said further pair of recognition sites, said further pair of recognition sites may flank a marker gene for enabling selection of cell clones for the presence or absence of said marker gene;
 wherein said further pair of two type IIs restriction endonuclease recognition sites are recognition sites of endonucleases different from the recognition sites of item (i) and item (iii) of  claim 1 .   
     
     
         8 . The system according to  claim 1 , wherein the cleavage sites of the recognition sites of item (i) are unique among the item (i) recognition sites of the set of n entry DNAs, and the cleavage sites of the recognition sites of item (iii) are unique among the item (iii) recognition sites within the set of n entry DNAs. 
     
     
         9 . The system according to  claim 1 , wherein the type IIs restriction endonuclease recognition sites of items (i) and (iii) are recognition sites of the same type IIs restriction endonuclease. 
     
     
         10 . The system according to  claim 1 , wherein the cleavage sites of the recognition sites of item (III) of all destination vectors are identical, and the cleavage sites of the recognition sites of item (I) of all destination vectors are non-identical. 
     
     
         11 . A method of producing a nucleic acid construct of interest from at least m nucleic acid fragment constructs numbered 1 to m, m being an integer of at least 3;
 said method comprising the following steps (A) to (C):   (A) providing said m nucleic acid fragment constructs, each of said m nucleic acid fragment constructs comprising in this order:
 (i′) a type IIs restriction endonuclease recognition site of the upstream cleavage site of item (ii′); 
 (ii′) a sequence segment of said nucleic acid construct of interest, said sequence segment comprising, in this order, an upstream cleavage site of the recognition site of item (i′), a core portion of the sequence segment, and a downstream cleavage site of the recognition site of the following item (iii′), and 
 (iii′) a type IIs restriction endonuclease recognition site of said downstream cleavage site of item (ii′); 
 the downstream cleavage sites of nucleic acid fragment constructs 1 to m−1 are complementary to the upstream cleavage sites of nucleic acid fragment constructs 2 to m, respectively, 
 the downstream cleavage site of a nucleic acid fragment construct u, wherein u is an integer that is <m and at least 2, is complementary to the upstream cleavage site of the type IIs restriction endonuclease recognition site of item (ii′) of nucleic acid fragment 1; 
   (B) reacting nucleic acid fragment constructs 1 to s, wherein s is an integer <u, a destination vector and a linker in the presence of a type IIs restriction endonuclease recognising said type IIs restriction endonuclease recognition sites of items (i′) and (iii′) and items (I) and (III) of the destination vector defined below and in the presence a DNA ligase in reaction medium compatible with activity of said type IIs restriction endonuclease and said ligase for recombining and ligating, in the following order, the sequence segment(s) of item (ii′) of nucleic acid fragment constructs 1 to s and said linker into said destination vector;
 said destination vector comprising in this order: 
 (I) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof complementary to the upstream cleavage site of item (ii′) of nucleic acid fragment construct 1; 
 (II) a vector backbone comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III); 
 (III) the further cleavage site of a type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site, 
 said linker being as defined in  claim 3 , wherein cleavage site (b) of said linker is complementary to the downstream cleavage site of item (ii′) of nucleic acid fragment construct s, and wherein cleavage site (g) of said linker and the cleavage site of item (III) of the destination vector are complementary; and 
   (C) treating a mixture comprising the recombination product of step (B) and nucleic acid fragment construct(s) s+1 to m with a type IIs restriction endonuclease recognising said type IIs restriction endonuclease recognition sites of items (i′) and (iii′), a type IIs restriction endonuclease recognising said type IIs restriction endonuclease recognition sites of items (d) and (e) of the linker and a DNA ligase in a reaction medium compatible with activity of said type IIs restriction endonucleases and said ligase for inserting the sequence segments of item (ii′) of nucleic acid fragment constructs s+1 to m and optionally a further linker as defined in  claim 3  into the cleavage sites provided by items (c) and (f) of the linker used in step (B).   
     
     
         12 . The method according to  claim 11 , wherein the recognition sites of all nucleic acid fragment constructs of items (i′) and (iii′), the recognition sites of items (a) and (h) of the linker and the recognition sites of item (I) and (III) of the destination vector are recognition sites of the same type IIs restriction endonuclease. 
     
     
         13 . System for producing a nucleic acid construct of interest, said system comprising:
 a set of n entry DNAs numbered 1 to n, n being an integer of at least 3,   each of said n entry DNAs comprising in this order:   (i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;   (ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and   (iii) a cleavage site of a further type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site;   the cleavage sites of the type IIs restriction endonuclease recognition sites of item (iii) of entry DNAs 1 to n−1 are complementary to the cleavage sites of the type IIs restriction endonuclease recognition sites of item (i) of entry DNAs 2 to n, respectively;   all cleavages sites of item (i) are unique among said n entry DNAs, and all cleavage sites of item (iii) are unique among said n entry DNAs;   said system further comprising a destination vector comprising in this order:   (I) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;   (II) a vector backbone comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III);   (III) a further cleavage site of a type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site, and   (IV) optionally, a linker between the recognition sites of item (III) and item (I); said system further comprising a nucleic acid linker comprising in the following order:   (a) a type IIs restriction endonuclease recognition site;   (b) a cleavage site of said recognition site of item (a);   (c) a cleavage site of a further type IIs restriction endonuclease recognition site of the following item (d);   (d) a type IIs restriction endonuclease recognition site defining the cleavage site of item (c) and being a recognition site of a type IIs restriction endonuclease different from that of item (a);   (e) a type IIs restriction endonuclease recognition site, preferably of the same endonuclease as the recognition site of item (d);   (f) a cleavage site of said recognition site of item (e);   (g) a cleavage site of a further type IIs restriction endonuclease recognition site of the following item (h);   (h) a type IIs restriction endonuclease recognition site defining the cleavage site of item (g), preferably of the same endonuclease as the recognition site of item (a);
 said linker being capable of linking a cleavage site of item (iii) of one of a entry DNA numbered 1 to n, preferably of number 1 to n−1, to a cleavage site of item (III) of said destination vector. 
   
     
     
         14 . The system according to  claim 13 , comprising the same number n of said linkers as the system comprises entry DNAs, said linkers being numbered 1 to n,
 wherein all linkers have the same cleavage site (g) that is complementary to the cleavage site of item (III) of said destination vector for linking each linker to the recognition site of item (III) of said destination vector, and   wherein each of said n linkers has a different cleavage site (b) that is complementary to the cleavage site of item (iii) of one of said n entry DNAs.   
     
     
         15 . The system according to  claim 14 , said system further comprising n different destination vectors, each destination vector being defined by items (I) to (IV) and having the same cleavage site of item (III) that is complementary to the cleavage site (g) of all linkers,
 each of said destination vectors having a different cleavage site if item (I) that is complementary to the cleavage site of item (i) of one of said n entry DNAs.   
     
     
         16 . System for producing a nucleic acid construct of interest, said system comprising:
 a set of n destination vectors (“destination vectors M”), n being an integer of at least 2, preferably at least 3,   each of said n destination vectors M comprising in the following order:   (I′) a type IIs restriction endonuclease recognition site defining the cleavage site of item (II′);   (II′) the cleavage site of said recognition site of item (I′);   (III′) a cleavage site of said recognition site of the following item (IV′);   (IV′) a further type IIs restriction endonuclease recognition site defining the cleavage site of item (III′) and being a different recognition site of a type IIs restriction endonuclease from that of item (I′);   (V′) a vector backbone comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of item and (IV′) and the following item (VI′);   (VI′) a further type IIs restriction endonuclease cleavage site;   (VII′) a type IIs restriction endonuclease recognition site of the cleavage site of item (VI′) and   (VIII′) optionally, an insert between the recognition sites of item (VII′) and item (I′); and   a set of n linkers M, n being as defined above, each linker M comprising in the following order:   (a′) a type IIs restriction endonuclease recognition site defining the cleavage site of item (b′);   (b′) the cleavage site of said recognition site of item (a′);   (c′) a cleavage site of a further type IIs restriction endonuclease recognition site of item (d′), said cleavage site having the same sequence of nucleotides as the cleavage site of item (b′);   (d′) the type IIs restriction endonuclease recognition site defining the cleavage site of item (c′) and being a different recognition site of a type IIs restriction endonuclease different from that of item (a′);   (e′) a further cleavage site of a type IIs restriction endonuclease recognition site of the following item (f′);   (f′) the type IIs restriction endonuclease recognition site defining the cleavage site of item (e′), that is preferably a recognition site of the same endonuclease as the recognition site of item (a′);   wherein   the cleavage sites (VI′) of all n destination vectors M are identical;   the cleavage sites (e′) of all n linkers M are identical;   the cleavage site of item (VI′) of each destination vector M is complementary to the cleavage site of item (e′) of each linker M for allowing annealing of single-stranded overhangs produced by the type IIs restriction endonuclease recognising recognition sites (VII′) and (f′);   the cleavage sites of items (II′) and (III′) within each destination vector M have the same sequence of nucleotides and may overlap such that one and the same sequence of nucleotides provides the cleavage site of items (II′) and that of item (III′); and   the cleavage sites of items (b′) and (c′) within each linker M have the same sequence of nucleotides and may overlap such that one and the same sequence of nucleotides provides the cleavage site of items (b′) and that of item (c′); and   the cleavage site (II′) of each destination vector M is unique among the cleavage sites (II′) of the set of n destination vectors M such that there are n different cleavage sites (II′), wherein for each of said n different cleavage sites (II′), there is a linker M having a cleavage site (b′) of identical nucleotide sequence among the set of n linkers M.   
     
     
         17 . The system according to  claim 16 , wherein
 (α) the recognition sites of items (a′) and (f′) of all n linkers M are recognition sites of the same type IIs restriction endonuclease;   (β) the recognition sites of items (d′) of all n linkers M are recognition sites of the same type IIs restriction endonuclease;   wherein the recognition sites of item (α) are different recognitions sites from those of item (β).   
     
     
         18 . The system according to  claim 16 , wherein
 (γ) the recognition sites of items (I′) and (VII′) of all n destination vectors M are recognition sites of the same type IIs restriction endonuclease;   (δ) the recognition sites of items (IV′) of all n destination vectors M are recognition sites of the same type IIs restriction endonuclease;   wherein the recognition sites of item (γ) are different recognitions sites from those of item (δ).   
     
     
         19 . The system according to  claim 16 , wherein
 the recognition sites of items (VII′) and (I′) of destination vectors M and of items (a′) and (f′) of the linkers M are recognition sites of the same type IIs restriction endonuclease;   the recognition sites of items (IV′) of destination vectors M and of items (d′) of the linkers M are recognition sites of the same type IIs restriction endonuclease.   
     
     
         20 . The system according to  claim 16 , comprising:
 a set of z entry DNAs numbered 1 to z, z being an integer of at least 2, preferably an integer of at least 3,   each of said z entry DNAs comprising in this order:   (i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;   (ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and   (iii) a cleavage site of a further type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site;   wherein   the cleavage site of item (i) of each entry DNA is complementary to the cleavage site of item (II′) of one of the n destination vectors M for allowing annealing of single-stranded overhangs produced by the type IIs restriction endonuclease recognising recognition sites of items (i) and (I′),   the recognition sites of item (i) of all z entry DNAs are preferably recognition sites of the same type IIs restriction endonuclease as the recognition sites of item (I′) and (VII′);   the cleavage site of item (iii) of each entry DNA is complementary to the cleavage sites of item (b′) of one of the n linkers M for allowing annealing of single-stranded overhangs produced by the type IIs restriction endonuclease recognising recognition sites of items (iii) and (a′),   the recognition sites of item (i) are recognition sites of the same type IIs restriction endonuclease as the recognition sites of item (a′) and (f′); and   the recognition sites of items (i) and (iii) of all z entry DNAs are recognition sites of the same type IIs restriction endonuclease.   
     
     
         21 . The system according to  claim 16 , further comprising a set of n destination vectors (“destination vectors P”), wherein n is as defined in  claim 1 ,
 each of said n destination vectors P comprising in the following order: 
 (I″) a type IIs restriction endonuclease recognition site defining the cleavage site of item (II″); 
 (II″) the cleavage site of said recognition site of item (I″); 
 (III″) a cleavage site of said recognition site of the following item (IV″); 
 (IV″) a further type IIs restriction endonuclease recognition site defining the cleavage site of item (III″) and being a different recognition site of a type IIs restriction endonuclease from that of item (I″); 
 (V″) a vector backbone comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of item and (IV″) and the following item (VI″); 
 (VI″) a further type IIs restriction endonuclease cleavage site; 
 (VII″) a type IIs restriction endonuclease recognition site of the cleavage site of item (VI″), preferably of the same endonuclease as the recognition site of item (I″) and 
 (VIII″) optionally, an insert between the recognition sites of item (VII″) and item (I″); and 
 a set of n linkers P, each linker P comprising in the following order: 
 (a″) a type IIs restriction endonuclease recognition site defining the cleavage site of item (b″); 
 (b″) the cleavage site of said recognition site of item (a″); 
 (c″) a cleavage site of a further type IIs restriction endonuclease recognition site of item (d″), said cleavage site having the same nucleotide sequence as the cleavage site of item (b″); 
 (d″) the type IIs restriction endonuclease recognition site defining the cleavage site of item (c″) and being a different recognition site of a type IIs restriction endonuclease from that of item (a″); 
 (e″) a further cleavage site of a type IIs restriction endonuclease recognition site of the following item (f″); 
 (f″) the type IIs restriction endonuclease recognition site defining the cleavage site of item (e″), that is preferably a recognition site of the same endonuclease as the recognition site of item (a″); 
 wherein 
 the cleavage sites (VI″) of all n destination vectors P are identical; 
 the cleavage sites (e″) of all n linkers P are identical; 
 the cleavage site of item (VI″) of each destination vector P is complementary to the cleavage site of item (e″) of each linker P for allowing annealing of single-stranded overhangs produced by the type IIs restriction endonuclease recognising recognition sites (VII″) and (f″); 
 the cleavage sites of items (II″) and (III″) within each destination vector P have the same sequence of nucleotides and may overlap such that one and the same sequence of nucleotides provides the cleavage site of item (II″) and the cleavage site of item (III″); 
 the cleavage sites of items (b″) and (c″) within each linker P have the same sequence of nucleotides and may overlap such that one and the same sequence of nucleotides provides the cleavage site of items (b″) and the cleavage site of item (c″); and 
 for each of said n different cleavage sites (b′) or (II′), there is a destination vector P having a cleavage site (II″) of identical nucleotide sequence as the nucleotide sequence of cleavage sites (b′) or (II′); and 
 for each of said n different cleavage sites (b′) or (II′), there is a linker P having a cleavage site (b″) of identical nucleotide sequence as the nucleotide sequence of cleavage sites (b′) or (II′). 
 
     
     
         22 . The system according to  claim 21 , wherein
 the recognition sites of items (I″) and (VII″) of all n destination vectors P are recognition sites of the same type IIs restriction endonuclease; and   the recognition sites of items (IV″) of all n destination vectors P are recognition sites of the same type IIs restriction endonuclease but different from the recognition sites of items (I″) and (VII″).   
     
     
         23 . The system according to  claim 21 , wherein
 the recognition sites of items (I″), (IV′), (d′), (a″) and (f″) are recognition sites of the same type IIs restriction endonuclease;   the recognition sites of items (IV″), (I′), (VII′), (a′) and (f′) are recognition sites of the same type IIs restriction endonuclease.   
     
     
         24 . (canceled)