Polynucleotide shuffling method

A method for preparing a Tile vector comprising a selectable marker and a coding polynucleotide sequence immediately preceded and followed by a type IIs recognition sequence, wherein the preceding and following recognition sequences are recognized by a same type IIs restriction enzyme, but have an opposite orientation,

• wherein the Tile vector can be cleaved using a type IIs restriction enzyme recognizing the preceding and following recognition sites resulting in the release of the coding polynucleotide sequence having at its respective ends known overhang sequences, the released coding polynucleotide sequence lacking the preceding and following type IIs recognition sequences;
• the method comprising:
• a) providing an initial coding polynucleotide and extending the respective ends of the polynucleotide with first and second terminal sequences wherein each of the terminal sequences comprises:
• i. a coding extension sequence consisting of one or more sets of three nucleotides, which is added adjacent to the respective end of and in frame with the open reading frame of the initial coding polynucleotide;
• ii. a first type IIs recognition sequence adjacent to the coding extension sequence wherein the first recognition sequence is oriented such that a type IIs restriction enzyme recognizing the first recognition site can cleave within the coding extension sequence generating an overhang and wherein the first type IIs recognition sequences of the first and second terminal sequences are recognized by a same type IIs enzyme, but have an opposite orientation;
• iii. a spacer sequence adjacent to or within the first type IIs recognition sequence;
• iv. a second type IIs recognition sequence adjacent to the spacer sequence wherein the second recognition sequence is oriented such that a type IIs restriction enzyme recognizing the second recognition sequence can cleave the spacer sequence to generate a spacer overhang and wherein the second type IIs recognition sequence is not recognized by a type IIs enzyme recognizing the first type IIs recognition sequence;
• v. a tail sequence of sufficient length in order to allow binding of a type IIs restriction enzyme to the second recognition sequence;
• b) providing a receiving vector comprising a first nucleotide sequence comprising a selectable marker positioned between first and second type IIs recognition sequences, such that the vector can be cleaved using type IIs recognition enzymes recognizing the first and second type IIs recognition sequences to form:
• i. a stuffer sequence comprising the first and second type IIs recognition sequences;
• and
• ii. a selectable vector fragment comprising the selectable marker but lacking the first and second type IIs recognition sequences and having non-complementary terminal overhangs, wherein one overhang is complementary to the spacer overhang obtained after cleaving the first terminal sequence using a type IIs recognition enzyme recognizing the second type IIs recognition sequence of the first terminal sequence, while the other overhang sequence is complementary to the spacer overhang obtained by cleaving the second terminal sequence using a type IIs restriction enzyme recognizing the second type IIs recognition sequence of the second terminal sequence;
• and
• c) incubating a mixture, wherein the mixture comprises:
• i. an extended initial coding polynucleotide of a);
• ii. a receiving vector of b);
• iii. type IIs restriction enzymes recognizing the second type IIs recognition sequences of the terminal sequences of the extended initial coding polynucleotide;
• iv. type IIs restriction enzymes recognizing the first and second type IIs recognition sequences of the receiving vector;
• and
• v. a DNA ligase.