Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids

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 (SS...

Full description

Saved in:

Bibliographic Details
Published in:	New biotechnology Vol. 72; no. C; pp. 22 - 28
Main Authors:	Jia, Huiyong, Couto-Rodriguez, Ricardo, Johnson, Sharon, Medina, Stephanie, Novillo, Brianna, Huynh, Peter, Kim, Matt, Cooper, Cade, Michalik, Meagan, Siew, Benjamin, Turesson, Elise, Maupin-Furlow, Julie A.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 25-12-2022 Elsevier
Subjects:	Amino acid exchange BASIC BIOLOGICAL SCIENCES Biochemistry & Molecular Biology Biotechnology & Applied Microbiology DNA Method Mutagenesis, Site-Directed Mutation PCR Plasmids - genetics Polymerase Chain Reaction - methods Site-directed mutagenesis Substitution mutation ss Apr rrPCR SSPER PAGE Nvr Method Substitution mutation 6-FAM LB Site-directed mutagenesis Amino acid exchange SDM PCR CFUs
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	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. •Two new methods are reported for the site-directed mutagenesis of large plasmids.•The methods are highly accurate, allowing for screening by DNA sequencing.•The procedures are cost-effective, requiring only two primers and two enzymes.•Mutations can be generated in single and pairwise combinations a single day.•The strategy is straightforward and easily implemented by entry level researchers.
Bibliography:	FG02-05ER15650; 5R01GM057498 National Institutes of Health (NIH) USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division (CSGB)
ISSN:	1871-6784 1876-4347
DOI:	10.1016/j.nbt.2022.08.004