US 5,512,463 AGranted

Enzymatic inverse polymerase chain reaction library mutagenesis

AssigneeEli Lilly and Company

InventorWillem P. C. Stemmer

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Abstract

This invention discloses a method for generating a recombinant library by introducing one or more changes within a predetermined region of double-stranded nucleic acid, comprising providing a first primer population and a second primer population, each of the populations having a variable base composition at known positions along the primers, the primers incorporating a class IIS restriction enzyme recognition sequence, being capable of directing change in the nucleic acid sequence and being substantially complementary to the double stranded nucleic acid to permit hybridization thereto. The method additionally comprises hybridizing the first and second primer populations to opposite strands of the double stranded nucleic acid to form a first pair of primer-templates oriented in opposite directions, performing enzymatic inverse polymerase chain reaction to generate at least one linear copy of the double stranded nucleic acid incorporating the change directed by the primers, cutting the double stranded nucleic acid copy with a class IIS restriction enzyme to form a restricted linear nucleic acid molecule containing the change, joining termini of the restricted linear nucleic acid molecule to produce double-stranded circular nucleic acid and introducing the nucleic acid into compatible host cells. A method is additionally provided for generating a recombinant library using wobble-base mutagenesis.

Claims27

Dependent claims

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.

9 more elements

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).

12 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/252,057Filed 1 Jun 1994 · continuation
Priority: 26 Apr 1991
Granted: 30 Apr 1996
Term ends: Not estimatedThe source records don’t support an estimate.

Classification7 codes

C12N 15/1093
Isolating an individual clone by screening libraries: general methods of preparing gene libraries, not provided for in other subgroups
C12N 15/102
Processes for the isolation, preparation or purification of DNA or RNA: mutagenizing nucleic acids
C12Q 1/6844
Involving nucleic acids: nucleic acid amplification reactions
C12Q 1/6853
Nucleic acid amplification reactions: using modified primers or templates