US 2006/0134624 A1 · Polymerase-based protocols for the introductions of deletions and insertions

Abstract

The invention relates to primers, libraries of primer, kits and methods for site-specific in vitro mutagenesis comprising: (a) cloning a parental polynucleotide into a vector comprising a cloning site, thereby obtaining a cloned product; (b) denaturing the cloned product, thereby obtaining a single-stranded polynucleotide template; (c) hybridizing at least one mutagenized oligonucleotide primer to the single-stranded polynucleotide template, thereby obtaining a first heteroduplex; (d) subjecting the first heteroduplex to linear amplification, thereby obtaining amplified products; (e) reacting the amplified products with a ligase, thereby obtaining ligated products; (f) denaturing the ligated products, thereby obtaining single stranded mutated polynucleotides; (g) hybridizing the single stranded mutated polynucleotides with a second oligonucleotide primer thereby obtaining second hybridized complexes; (h) copying the second hybridized complex and ligating the double stranded product thereof; thereby obtaining a circular double stranded mutated polynucleotide; (i) transforming the double-stranded mutated polynucleotide into a bacterial host, thereby obtaining transformants.

Claims43

Dependent claims

A method for polynucleotide mutagenesis comprising:

(a) cloning a parental polynucleotide into a vector comprising a cloning site, thereby obtaining a cloned product;
(b) denaturing the cloned product, thereby obtaining a single-stranded polynucleotide template;
(c) hybridizing at least one mutagenized oligonucleotide primer to the single-stranded polynucleotide template, thereby obtaining a first heteroduplex;
(d) extending the first heteroduplex with a polymerase, thereby obtaining an extended product;
(e) reacting the extended product with a ligase, thereby obtaining ligated product;
(f) denaturing the ligated product, thereby obtaining a closed single stranded mutated polynucleotide;
(g) optionally repeating steps (c)-(f);
(h) hybridizing the single stranded mutated polynucleotides with a second oligonucleotide primer thereby obtaining second hybridized complexes;
(i) copying the second hybridized complex and ligating the double stranded product thereof, thereby obtaining a circular double stranded mutated polynucleotide;
and
(j) transforming the double-stranded mutated polynucleotide into a bacterial host, thereby obtaining transformants.

8 more elements

Dependencies are inferred from the claim text.

Polymerase-based protocols for the introductions of deletions and insertions

Life of this patent

Polymerase-based protocols for the introductions of deletions and insertions

Life of this patent

Abstract

Claims43