{"doi":"10.1016/j.nbt.2022.08.004","title":"Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids","abstract":"Advances are needed in the site-directed mutagenesis of large plasmids for protein structure-function studies, as current methods are often inefficient, complicated and time-consuming. Here two new methods are reported that overcome these difficulties, namely the single primer extension reaction (SSPER) strategy that reaches 100% efficiency and the reduce recycle PCR (rrPCR) method that is advantageous in generating single and pairwise combinations of mutations. Both methods are distinguished from current technologies by the addition of a step that easily removes the oligonucleotide primer(s) after the first reaction, thus allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). High efficiency of the methods is demonstrated by generating single and paired combinations of the 11 site-directed mutations targeted on 5 different plasmid DNA templates ranging from 10 to 12 kb and 57-60% GC-content at a rate of 50-100%. Overall, the methods are demonstrated to be (i) highly accurate, allowing for screening of plasmids by DNA sequencing, (ii) streamlined to generate the mutations within a single day, (iii) cost-effective in requiring only two primers and two enzymes (DpnI and a proofreading DNA polymerase), (iv) straightforward in primer design, (v) applicable for both large and small plasmids, and (vi) easily implemented by entry level researchers.","journal":"New Biotechnology","year":2022,"id":266675,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":9,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9518,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":767322,"name":"Ricardo L. Couto-Rodríguez","orcid":"0000-0002-4222-9538","position":1,"is_corresponding":false},{"id":927238,"name":"Sharon Johnson","orcid":null,"position":2,"is_corresponding":false},{"id":927239,"name":"Stephanie Medina","orcid":null,"position":3,"is_corresponding":false},{"id":927240,"name":"Brianna Novillo","orcid":null,"position":4,"is_corresponding":false},{"id":244259,"name":"Peter Huynh","orcid":null,"position":5,"is_corresponding":false},{"id":927241,"name":"Matt Kim","orcid":null,"position":6,"is_corresponding":false},{"id":927242,"name":"Cade Cooper","orcid":null,"position":7,"is_corresponding":false},{"id":927243,"name":"Meagan Michalik","orcid":null,"position":8,"is_corresponding":false},{"id":927244,"name":"Benjamin Siew","orcid":null,"position":9,"is_corresponding":false},{"id":927245,"name":"Elise Turesson","orcid":null,"position":10,"is_corresponding":false},{"id":345489,"name":"Julie A. Maupin‐Furlow","orcid":"0000-0001-6105-0923","position":11,"is_corresponding":false},{"id":483882,"name":"Huiyong Jia","orcid":null,"position":0,"is_corresponding":true}],"reference_count":22,"raw_metadata":null,"created_at":"2026-07-19T00:26:58.102271Z","pmid":"36007808","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":[]}