Novel methods of directed evolution
AssigneeRensselaer Polytechnic Institute
InventorJohn C. Salerno
Life of this patent
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Abstract
Methods for generating chimeric polynucleotides by directed evolution are described. In the methods, splice points of interest are identified within the polynucleotides of a basis set of polynucleotides, preferably through the use of an algorithm that defines the number of splice points and selects the splice points, either by random selection or using information regarding alignment of the polynucleotides. The algorithms can include additional factors, including a definition of a desired distance between splice points, and/or weighing factors to bias selection of splice points. Chimeric polynucleotides are generated using primers (e.g., double primers or non-overlapping primers) and polymerase chain reaction or combinatorial strategies.
Claims41
A method for generating chimeric polynucleotides, comprising:
- a) providing a basis set of polynucleotides, wherein the basis set comprises two or more different polynucleotides;
- b) identifying splice points within the polynucleotides of the basis set, wherein each polynucleotide in the basis set has the same number of splice points;
- c) generating oligonucleotide double primer sets for each splice point, wherein each double primer in a set comprises a “pre” region joined to and followed immediately by a “post” region, and wherein the “pre” region comprises an oligonucleotide primer for a splice point in one polynucleotide in the basis set, and the “post” region comprises the complement of an oligonucleotide primer for that splice point in another polynucleotide in the basis set, and wherein the set of double primers includes double primers comprising all possible combinations of pre and post regions for each splice point;
- d) using the double primer sets in polymerase chain reaction to amplify combinations of fragments;
- thereby generating a multitude of chimeric polynucleotides, wherein each chimeric polynucleotide comprises a fragment from at least two of the polynucleotides in the basis set.
A method for generating chimeric polynucleotides, comprising:
- a) providing a basis set of polynucleotides, wherein the basis set comprises two or more different polynucleotides;
- b) identifying splice points of interest within the polynucleotides of the basis set, wherein each polynucleotide in the basis set has the same number of splice points, and wherein the splice points divide each polynucleotide into M consecutive fragments in a correct order;
- c) generating non-overlapping oligonucleotides for each fragment of the M fragments for each polynucleotide in the basis set;
- d) ligating oligonucleotides corresponding to consecutive fragments in the correct order;
- e) selecting correctly ordered combinations of fragments;
- thereby generating a multitude of correctly ordered chimeric polynucleotides, wherein each chimeric polynucleotide comprises a fragment from each of the polynucleotides in the basis set.
Dependencies are inferred from the claim text.