Dataset Information

A python script to design site-directed mutagenesis primers.

ABSTRACT: Primer design is essential to conduct whole plasmid site-directed mutagenesis for protein study. Traditionally, primers of mutagenesis are designed manually that is time-consuming and fallible. Here, we present a Python script for searching primers by presetting parameters of nucleotide composition and percentage of guanine-cytosine (GC content). The running results showed that the script is able to search primers with mutations of target residue automatically. This script may facilitate primer design for whole plasmid site-directed mutagenesis and aid protein mutant construction.

SUBMITTER: Bi C

PROVIDER: S-EPMC7096704 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A python script to design site-directed mutagenesis primers.

Bi Chuyun C Huang Xiaofang X Tang Dan D Shi Yuanyuan Y Zhou Lixiang L Hu Yunzhuo Y Chen Xuanyang X Qi Shiqian S Lin Shiqiang S

Protein science : a publication of the Protein Society 20191227 4

Primer design is essential to conduct whole plasmid site-directed mutagenesis for protein study. Traditionally, primers of mutagenesis are designed manually that is time-consuming and fallible. Here, we present a Python script for searching primers by presetting parameters of nucleotide composition and percentage of guanine-cytosine (GC content). The running results showed that the script is able to search primers with mutations of target residue automatically. This script may facilitate primer ...[more]

PMID: 31855299

Similar Datasets

Project description:BackgroundSite-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have been developed, methods that increase efficiency and versatility of this process remain highly desired.MethodWe present a versatile and simple method to efficiently introduce a variety of mutation schemes using Gibson-assembly but without the need to design uniquely designated Gibson primers. Instead, we explore the re-use of standard SDM primers (completely overlapping in sequence) in combination with regular primers (~ 25 bps long) for amplification of fragments flanking the site of mutagenesis. We further introduce a rapid amplification step of the Gibson-assembled product for analysis and quality control, as well as for ligation, or re-ligation at instances the process fails (avoiding expenditure of added Gibson reaction mixtures).ResultsWe first demonstrate that standard SDM primers can be used with the Gibson assembly method and, despite the need for extensive digestion of the DNA past the entire primer sequence, the reaction is attainable within as short as 15 min. We also find that the amount of the assembled Gibson product is too low to be visualized on standard agarose gel. Our added amplification step (by use of the same short primers initially employed) remedies this limitation and allows to resolve whether the desired Gibson-assembled product has been obtained on agarose gel or by sequencing of amplicons. It also provides large amounts of amplicons for subsequent ligations, bypassing the need to re-employ Gibson mixtures. Lastly, we find that our method can easily accommodate SDM primers with degenerate sequences.ConclusionWe employ our alternative approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). Importantly, our approach involves the re-use of SDM primers from our primer-inventory. Our scheme thereby reduces the need (and time and money) to design and order new custom Gibson-primers. Together, we provide a simple and versatile protocol that spans only 4 days (including the added amplification step), requires minimal primer sets and provides very high yields and success rates (> 98%).

Dataset Information

A python script to design site-directed mutagenesis primers.

Publications

A python script to design site-directed mutagenesis primers.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets