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%.
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.
Claim 2
Dependent Claim
126 words
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.
Claim 3
Dependent Claim
19 words
A method of claim 1 wherein the hydrolysis of the uracil-containing strand results from in vitro exposure to glycosylase.
Claim 4
Dependent Claim
32 words
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.
Claim 5
Dependent Claim
19 words
A method of claim 1 wherein the uracil-containing template is produced in E. coli dut - ung - cells.