{"doi":"10.1073/pnas.82.2.488","title":"Rapid and efficient site-specific mutagenesis without phenotypic selection.","abstract":"<jats:p>Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.</jats:p>","journal":"Proceedings of the National Academy of Sciences","year":1985,"id":8321,"datarank":27.817344800120047,"base_score":8.898911905794401,"endowment":8.898911905794401,"self_citation_contribution":1.3348367858691603,"citation_network_contribution":26.482508014250886,"self_endowment_contribution":1.3348367858691603,"citer_contribution":26.482508014250886,"corpus_percentile":96.8,"corpus_rank":2361,"citation_count":7323,"citer_count":199,"citers_with_citation_signal":199,"citers_with_endowment":199,"datacite_reuse_total":0,"is_dataset":false,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"1985-01-01","authors":[{"id":73052,"name":"Thomas A. Kunkel","orcid":"0000-0002-9900-1788","position":1,"is_corresponding":false},{"id":73051,"name":"T A Kunkel","orcid":null,"position":0,"is_corresponding":true}],"reference_count":29,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-03-01T18:20:47.508186Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}