{"doi":"10.1152/ajpcell.00517.2004","title":"A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study","abstract":"<jats:p> PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology. </jats:p>","journal":"American Journal of Physiology-Cell Physiology","year":2005,"id":31747,"datarank":3.760674403133935,"base_score":4.1588830833596715,"endowment":4.1588830833596715,"self_citation_contribution":0.6238324625039509,"citation_network_contribution":3.136841940629984,"self_endowment_contribution":0.6238324625039509,"citer_contribution":3.136841940629984,"corpus_percentile":null,"corpus_rank":null,"citation_count":63,"citer_count":56,"citers_with_citation_signal":51,"citers_with_endowment":51,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":169493,"name":"Jianjie Ma","orcid":null,"position":1,"is_corresponding":false},{"id":169492,"name":"Jae-Kyun Ko","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"base_score":4.1588830833596715,"endowment":4.1588830833596715,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"15659713","pmcid":null,"openalex_id":"https://openalex.org/W2113333529","authors":[],"funders":[{"funder_name":"NIDDK NIH HHS","grant_id":"R01 DK-51770","title":null},{"funder_name":"NIA NIH HHS","grant_id":"R01 AG-15556","title":null},{"funder_name":"NCI NIH HHS","grant_id":"R01 CA-95739","title":null},{"funder_name":"NHLBI NIH HHS","grant_id":"R01 HL-69000","title":null}],"total_grants":4,"fwci":1.5161,"citation_percentile":0.81297724,"influential_citations":1,"citation_trend":[{"year":2012,"count":5},{"year":2013,"count":3},{"year":2014,"count":2},{"year":2015,"count":4},{"year":2016,"count":2},{"year":2017,"count":3},{"year":2018,"count":2},{"year":2019,"count":4},{"year":2020,"count":1},{"year":2023,"count":1},{"year":2024,"count":2},{"year":2025,"count":1}],"oa_status":"closed","license":null,"oa_locations":[{"url":"https://journals.physiology.org/doi/pdf/10.1152/ajpcell.00517.2004","host_type":"publisher"},{"url":"https://doi.org/10.1152/ajpcell.00517.2004","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/15659713","host_type":"repository"}],"fields_of_study":["RNA and protein synthesis mechanisms","Molecular Biology Techniques and Applications","CRISPR and Genetic Engineering","Biology","Medicine","Base Sequence","Cell Physiological Phenomena","DNA Primers","Deoxyribonucleases, Type II Site-Specific","Multigene Family","Mutagenesis, Site-Directed","Polymerase Chain Reaction"],"mesh_terms":["Base Sequence","Cell Physiological Phenomena","Multigene Family","Deoxyribonucleases, Type II Site-Specific","Polymerase Chain Reaction","Mutagenesis, Site-Directed","DNA Primers"],"keywords":["Mutagenesis","Site-directed mutagenesis","Gene","Restriction site","Biology","Computational biology","Restriction enzyme","Restriction digest","Protein engineering","Directed mutagenesis","Genetics","Mutation","Enzyme","Biochemistry","Mutant"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-06-09T08:27:49.275717Z","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,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}