US 5,514,568 A · Enzymatic inverse polymerase chain reaction

Abstract

The invention is directed to a method of introducing at least one predetermined change in a nucleic acid sequence of a double-strand DNA. The method includes: (a) providing a first primer and a second primer capable of directing said predetermined change in said nucleic acid sequence, said first and second primers comprising a nucleic acid sequence substantially complementary to said double-stranded DNA so as to allow hybridization, a class IIS restriction enzyme recognition sequence and cleavage sites; (b) hybridizing said first and second primers to opposite strands of said double-stranded DNA to form a first pair of primer-templates oriented in opposite directions; (c) extending said first pair of primer-templates to create double-stranded molecules; (d) hybridizing said first and second primers at least once to said double-stranded molecules to form a second pair of primer-templates; (e) extending said second pair of primer-templates to produce double-stranded linear molecules terminating with class IIS restriction enzyme recognition sequences; and (f) restricting said double-stranded linear molecules with a class IIS restriction enzyme to form restricted linear molecules containing said change in said nucleic acid sequence. Also provided is a method of producing at least two changes located at one or more positions within a nucleic acid sequence. Synthetic primers for producing such changes are provided as well.

Claims18

Dependent claims

A method of introducing at least one predetermined change in a circular double-stranded DNA sequence comprising:

(a) providing a first primer and a second primer wherein said first and second primers each comprise in a 5' to 3' orientation a class IIS restriction enzyme recognition sequence, a class IIS restriction enzyme cleavage site associated with said class IIS restriction enzyme recognition sequence, said predetermined change to be incorporated into said double-stranded circular DNA sequence, and a sequence substantially complementary to said double-stranded DNA;
(b) hybridizing said first and second primers to opposite strands within the same region of said double stranded DNA sequence in the same reaction vessel;
(c) performing at least one cycle of a polymerase chain reaction to produce double-stranded linear molecules terminating with said class IIS restriction enzyme recognition sequence and including said class IIS restriction enzyme cleavage site and said predetermined change;
(d) cleaving said double-stranded linear molecules with at least one class IIS restriction enzyme at said class IIS restriction enzyme cleavage sites to produce overhanging termini that are complementary to one other;
and
(e) ligating said overhanging termini to recircularize said double-stranded linear molecules containing said predetermined change.

3 more elements

A method of introducing at least two changes located at one or more positions within a circular double-stranded DNA sequence comprising:

(a) providing a first primer and a second primer population wherein said first and second primer populations each comprise in a 5' to 3' orientation a class IIS restriction enzyme recognition sequence, a class IIS restriction enzyme cleavage site associated with said class IIS restriction enzyme recognition sequence, said changes to be incorporated into said double-stranded circular DNA sequence, and a sequence substantially complementary to said double-stranded DNA;
(b) hybridizing said first and second primer populations to opposite strands of the same region of said double-stranded DNA sequence in the same reaction vessel;
(c) performing at least one cycle of a polymerase chain reaction to produce a population of double-stranded linear molecules terminating with said class IIS restriction enzyme recognition sequence and including said class IIS restriction enzyme cleavage site and said changes in said double-stranded DNA sequence;
(d) cleaving said double-stranded linear molecules with at least one class IIS restriction enzyme at said class IIS restriction enzyme cleavage sites to produce overhanging termini that are complementary to one other;
and
(e) ligating said overhanging termini to recircularize said double-stranded linear molecules containing said changes located at one or more positions within said nucleic acid sequence.

3 more elements

Dependencies are inferred from the claim text.

Enzymatic inverse polymerase chain reaction

Life of this patent

Enzymatic inverse polymerase chain reaction

Life of this patent

Abstract

Claims18