Enzymatic inverse polymerase chain reaction library mutagenesis

Claim 1

A method for generating a recombinant mutagenesis library by introducing one or more changes within a predetermined region of a double stranded circular DNA sequence, comprising the steps of: (a) providing a first primer population and a second primer population, eaeh of said primer populations comprising in a 5' to 3' orientation; (i) a class IIS recognition sequence, wherein said IIS recognition sequence is not complementary to the DNA sequence of the predetermined region of the double stranded circular DNA sequence to be mutagenized; (ii) a class IIS restriction enzyme cleavage site associated with said class IIS restriction enzyme recognition sequence; (iii) a region of variable base composition at predetermined positions in said primers; and (iv) a DNA sequence substantially complementary to said double stranded circular DNA sequence to allow hybridization thereto, wherein the DNA sequence immediately adjacent to said class IIS restriction enzyme cleavages site within said primer populations is not complementary to the DNA sequence of the predetermined region of the double stranded circular DNA sequence to be mutagenized; (b) denaturing said double stranded circular DNA sequence and hybridizing said first and second primer populations to opposite strands of said denatured double stranded circular DNA sequence to form a first pair of primer templates wherein said primers are oriented in opposite directions relative to one another and wherein said class IIS recognition sequence does not hybridize to the denatured double stranded circular DNA sequence; (c) performing extension, denaturing and hybridization steps of a polymerase chain reaction to generate at least one linear copy of said double stranded circular DNA sequence to incorporate said change directed by said primers; (d) cutting the linear copy of said double stranded circular DNA sequence of step (c) with a class IIS restriction enzyme to form a restricted linear DNA molecule containing said change to produce overhanging termini that are complementary to one another; (e) ligating intramolecularly said overhanging termini of said linear DNA molecule to recircularize said linear copy of said double stranded circular DNA sequence containing said change; and (f) introducing said recircularized copies of double stranded DNA sequence into compatible host cells, wherein a recombinant mutagenesis library is generated.

Claim 2

A method for generating a recombinant mutagenesis library by introducing one or more changes within a predetermined region of a double stranded circular DNA sequence, comprising the steps of: (a) providing a first primer population and a second primer population, eaeh of said primer populations comprising in a 5' to 3' orientation; (i) a class IIS recognition sequence, wherein said IIS recognition sequence is not complementary to the DNA sequence of the predetermined region of the double stranded circular DNA sequence to be mutagenized; (ii) a class IIS restriction enzyme cleavage site associated with said class IIS restriction enzyme recognition sequence; (iii) a region of variable base composition at predetermined positions in said primers; and (iv) a DNA sequence substantially complementary to said double stranded circular DNA sequence to allow hybridization thereto, wherein the DNA sequence immediately adjacent to said class IIS restriction enzyme cleavages site within said primer populations is not complementary to the DNA sequence of the predetermined region of the double stranded circular DNA sequence to be mutagenized; (b) denaturing said double stranded circular DNA sequence and hybridizing said first and second primer populations to opposite strands of said denatured double stranded circular DNA sequence to form a first pair of primer templates wherein said primers are oriented in opposite directions relative to one another and wherein said class IIS recognition sequence does not hybridize to the denatured double stranded circular DNA sequence; (c) performing extension, denaturing and hybridization steps of a polymerase chain reaction to generate at least one linear copy of said double stranded circular DNA sequence to incorporate said change directed by said primers; (d) cutting the linear copy of said double stranded circular DNA sequence of step (c) with a class IIS restriction enzyme to form a restricted linear DNA molecule containing said change to produce overhanging termini that are complementary to one another; (e) ligating intramolecularly said overhanging termini of said linear DNA molecule to recircularize said linear copy of said double stranded circular DNA sequence containing said change; and (f) introducing said recircularized copies of double stranded DNA sequence into compatible host cells, wherein a recombinant mutagenesis library is generated.

Claim 15

A method for improving polypeptide expression from a double stranded circular DNA sequence encoding polypeptide, comprising the steps of: (a) measuring polypeptide expression from said double-stranded DNA in a compatible host cell; (b) providing a first primer population and a second primer population, each of said primer populations comprising in a 5' to 3' orientation: (i) a class IIS recognition sequence, wherein said class IIS recognition sequence is not complementary to a DNA sequence of a predetermined region of the double stranded circular DNA sequence to be mutagenized; (ii) a class IIS restriction enzyme cleavage site associated with said class IIS restriction enzyme recognition sequence; (iii) a region of variable base composition at predetermined positions along said primers; and (iv) a sequence substantially complementary to said double stranded circular DNA sequence to allow hybridization thereto, wherein the DNA sequence immediately adjacent to said class IIS restriction enzyme cleavage site within said primer populations is not complementary to the DNA sequence of the predetermined region of the double stranded circular DNA sequence to be mutagenized; (c) denaturing said double stranded circular DNA sequence and hybridizing said first and second primer populations to opposite strands of said denatured double stranded circular DNA sequence to form a first pair of primer-templates wherein said primers are oriented in opposite directions relative to one another and wherein said class IIS recognition sequence does not hybridize to the denatured double stranded circular DNA sequence; (d) performing extension, denaturating and hybridization steps of a polymerase chain reaction to generate at least one linear copy of said double stranded circular DNA sequence incorporating said change directed by said primers; (e) cutting the linear copy of said double stranded circular DNA sequence of step (d) with a class IIS restriction enzyme to form a restricted linear DNA molecule containing said change to produce overhanging termini that are complementary to one another; (f) ligating intramolecularly said overhanging termini of said linear DNA molecule to recircularize said linear copy of said double stranded circular DNA sequence containing said change; (g) introducing said recircularized copies of double stranded DNA from step (f) into said host cells; (h) measuring polypeptide expression from said modified DNA of step (g) in said cells; and (i) identifying cells with polypeptide expression greater than the polypeptide expression measured in step (a).

Claim 16

A method for improving polypeptide expression from a double stranded circular DNA sequence encoding polypeptide, comprising the steps of: (a) measuring polypeptide expression from said double-stranded DNA in a compatible host cell; (b) providing a first primer population and a second primer population, each of said primer populations comprising in a 5' to 3' orientation: (i) a class IIS recognition sequence, wherein said class IIS recognition sequence is not complementary to a DNA sequence of a predetermined region of the double stranded circular DNA sequence to be mutagenized; (ii) a class IIS restriction enzyme cleavage site associated with said class IIS restriction enzyme recognition sequence; (iii) a region of variable base composition at predetermined positions along said primers; and (iv) a sequence substantially complementary to said double stranded circular DNA sequence to allow hybridization thereto, wherein the DNA sequence immediately adjacent to said class IIS restriction enzyme cleavage site within said primer populations is not complementary to the DNA sequence of the predetermined region of the double stranded circular DNA sequence to be mutagenized; (c) denaturing said double stranded circular DNA sequence and hybridizing said first and second primer populations to opposite strands of said denatured double stranded circular DNA sequence to form a first pair of primer-templates wherein said primers are oriented in opposite directions relative to one another and wherein said class IIS recognition sequence does not hybridize to the denatured double stranded circular DNA sequence; (d) performing extension, denaturating and hybridization steps of a polymerase chain reaction to generate at least one linear copy of said double stranded circular DNA sequence incorporating said change directed by said primers; (e) cutting the linear copy of said double stranded circular DNA sequence of step (d) with a class IIS restriction enzyme to form a restricted linear DNA molecule containing said change to produce overhanging termini that are complementary to one another; (f) ligating intramolecularly said overhanging termini of said linear DNA molecule to recircularize said linear copy of said double stranded circular DNA sequence containing said change; (g) introducing said recircularized copies of double stranded DNA from step (f) into said host cells; (h) measuring polypeptide expression from said modified DNA of step (g) in said cells; and (i) identifying cells with polypeptide expression greater than the polypeptide expression measured in step (a).