Circular site-directed mutagenesis

• US 5,932,419 A
• Filed: 04/17/1997
• Issued: 08/03/1999
• Est. Priority Date: 12/08/1995
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Children's Medical Center Corporation, Stratagene La Jolla California
• Inventors: Wright, Dowain A., Bauer, John C., Geha, Raif S., Braman, Jeffrey Carl
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/844,524
• Time in Patent Office: 838 Days
• Field of Search: 435/5, 435/6, 435/91.1, 435/91.2, 435/810, 435/183, 435/195, 435/196
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed. The double-stranded circular DNA intermediates is transformed in suitable competent host cells and closed circular double-stranded DNA corresponding to the parental template molecules, but containing the desired mutation or mutations of interest, may be conveniently recovered from the transformed cells. The invention also provide kits for site-directed mutagenesis in accordance with methods of the present invention.

Claims

1. A method of mutagenizing a selected DNA molecule, wherein said DNA molecule is double-stranded and circular, and said method comprises:

annealing a first mutagenic primer to a first strand of said DNA molecule and a second mutagenic primer to a second strand of said DNA molecule, wherein said first mutagenic primer comprises a region that is complementary to the second mutagenic primer and wherein said first and second mutagenic primers each contain at least one mutation site with respect to said DNA molecule, wherein the mutation site is located within the region that is complementary between said first and second mutagenic primers;

synthesizing by means of a linear cyclic amplification reaction a first mutagenized DNA strand comprising said first mutagenic primer and a second mutagenized DNA strand comprising said second mutagenic primer, wherein the first mutagenized DNA strand and the second mutagenized DNA strand form a double-stranded mutagenized circular DNA intermediate; and

digesting the non-mutagenized strands of said DNA molecule, wherein said digestion is mediated by a selection enzyme.

2. The method according to claim 1, wherein the selection enzyme is a restriction endonuclease.

3. The method according to claim 1, wherein the linear cyclic amplification reaction is catalyzed by Pfu DNA polymerase.

4. The method according to claim 1, wherein the first and second mutagenic primers are 5'"'"' phosphorylated.

5. The method according to claim 1, wherein the linear cyclic amplification reaction is repeated for less than 20 cycles.

6. The method according to claim 1, wherein the first and second mutagenic primers are completely complementary to each other.

7. The method according to claim 1, said method further comprising the steps,

annealing said first mutagenized DNA strand and the second mutagenized DNA strand so as to form a double-stranded mutagenized circular DNA intermediate, and

transforming a host cell with said double-stranded mutagenized circular DNA intermediate.