Process for site specific mutagenesis without phenotypic selection

• US 4,873,192 A
• Filed: 05/18/1988
• Issued: 10/10/1989
• Est. Priority Date: 02/17/1987
• Status: Expired due to Term

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Kunkel, Thomas A.
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): Seidman, S.
• Application Number:
  US07/196,779
• Time in Patent Office: 510 Days
• Field of Search: 435/6, 435/68, 435/91, 435/70, 435/172.1, 435/172.3, 435/317.1, 435/320, 536/27, 935/10, 935/16
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

Abstract

The present invention discloses several DNA mutagenesis processes using a DNA template containing several uracil residues in place of thymine, which can be applied without selection techniques to produce altered DNA sequences with approximately 10-fold greater efficiency than current methods of site-specific mutagenesis.

This template has relatively normal coding potential in the in vitro reactions typical of standard site-directed mutagenesis protocols but is not biologically active upon transfection into a wild type (i.e., ung⁺) E. coli host cell. Expression of a desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus favored. The procedure has been applied to mutations introduced via both obligonucleotides and error-prone polymerization. The inclusion of two additional simple treatment steps before transfection results in a site-specific mutation frequency approaching 100%.

Claims

1. A process for producing site-specific mutageneis in genes comprising the steps of:

(1) Separating the strands of a DNA molecule that encodes a peptide;

(2) Replacing thymine with uracil in one strand of the DNA from step (1);

(3) Preparing a single-stranded DNA fragment that contains one or more mismatched nucleotides and that will hybridize to the uracil-containing DNA strand prepared in step (2);

(4) Hybridizing the uracil-containing strand prepared in step (2) to the strand of DNA fragment prepared in step (3) to form a heteroduplex;

(5) Converting the heteroduplex formed in step (4) to a full-length double-stranded DNA molecule; and

(6) Hydrolyzing the uracil-containing strand by in vitro treatment with uracil-glycosylase or by inserting the product of step (5) into a ung+ E. coli.

2. A method of claim 1 wherein the hydrolysis of the uracil-containing strand results from in vitro exposure to glycosylase.

3. A method of claim 1 wherein the hydrolysis of the uracil-containing strand results from insertion of the full-length double-stranded DNA molecule of step (5) of claim 1 into a ung+ E. coli.

4. A method of claim 1 wherein the uracil-containing template is produced in E. coli dut^- ung^- cells.