scholarly journals A Simplified Gibson Assembly Method for Site Directed Mutagenesis Using Non-Gibson Primers

2021 ◽  
Author(s):  
Shunit Olszakier ◽  
Shai Berlin
Keyword(s):  
Dna Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Success Rates ◽  
Simple Method ◽  
Gibson Assembly ◽  
Assembly Method ◽  
Rapid Amplification ◽  
High Yields ◽  
Primer Sequence

Abstract Background: Site-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. Method: We present a versatile and simple method to efficiently introduce a variety of mutation schemes using the Gibson-assembly without the need for unique Gibson primers. The method entails use of standard SDM primers (shorter and completely overlapping in sequences in contrast to Gibson primers) that are separately employed with common primer (~25 bps long) for amplification of fragments flanking the site of mutagenesis, followed by rapid amplification of the Gibson-assembled product for added visualization and sequencing steps for ensuring high success rates.Results: We find that assembly of the fragments via the Gibson reaction mixture is attainable within as short as 15 minutes, despite the need for extensive digestion of the DNA (by exonuclease) past the entire SDM primer sequence (to expose non-clashing overlap between the fragments). We also find that the amount of the assembled Gibson product is too low to be visualized and assessed on standard agarose gel. We thereby introduce a short amplification step (by use of the same short primers initially employed) to 1) easily resolve whether the product (only the correct size can yield a product) has been obtained, and 2) for isolation of product for DNA-sequencing (to assess whether mutation(s) have been introduced). No other SDM method enables assessment of mutagenesis prior completion of the process. Conclusion: We employ our approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically in DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). The entire protocol spans only four days, requires minimal primers sets (as well as can be used with most in-house primers) and provides very high yields and success rates (>98%).

scholarly journals A simple method for site-directed mutagenesis using the polymerase chain reaction

1989 ◽  
Vol 17 (16) ◽  
pp. 6545-6551 ◽  
Author(s):  
Anne Hemsley ◽  
Norman Arnheim ◽  
Michael Dennis Toney ◽  
Gino Cortopassi ◽  
David J. Galas
Keyword(s):  
Polymerase Chain Reaction ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Chain Reaction ◽  
Simple Method ◽  
Polymerase Chain

scholarly journals A short 5'-flanking region mediates glucose repression of amylase gene expression in Drosophila melanogaster.

Genetics ◽  
1993 ◽  
Vol 134 (2) ◽  
pp. 507-515 ◽  
Author(s):  
C Magoulas ◽  
L Bally-Cuif ◽  
A Loverre-Chyurlia ◽  
B Benkel ◽  
D Hickey
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Glucose Repression ◽  
Essential Elements ◽  
Site Directed Mutagenesis ◽  
Deletion Analysis ◽  
Upstream Region ◽  
Directed Mutagenesis ◽  
Amylase Gene ◽  
Recombinant Gene

Abstract Expression of the alpha-amylase gene is highly repressed by dietary glucose in Drosophila melanogaster larvae. Here, we show that glucose repression is controlled by DNA sequences that are located upstream of the transcribed region. Recombinant gene constructions, in which the amylase promoter sequences were fused with the transcribed region of the Adh gene, were expressed in transgenic Drosophila larvae. The expression of ADH from the recombinant gene was shown to be subject to glucose repression. The function of potential regulatory cis-acting elements within the glucose responsive upstream region was examined by deletion analysis and by site-directed mutagenesis, coupled with expression assays in transformed larvae. The upstream deletion analysis showed that essential elements, both for overall activity and for glucose repression of the amylase gene, are located within a 109-bp region upstream of the transcription start site. Site-directed mutagenesis of these upstream sequences showed that the TATA motif, at position -31, and a novel 36-bp element, at position -109, were necessary for full activity of the amylase promoter. None of the introduced mutations resulted in loss of glucose responsiveness. These results indicate that glucose repression, in Drosophila, is mediated by transcriptional mechanisms that involve multiple, functionally redundant DNA elements.

scholarly journals DNA Gap Repair-Mediated Site-Directed Mutagenesis is Different from Mandecki and Recombineering Approaches

2018 ◽  
Author(s):  
George T. Lyozin ◽  
Luca Brunelli
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Products ◽  
E Coli ◽  
Health And Disease ◽  
First Time ◽  
Disease Understanding

AbstractSite-directed mutagenesis allows the generation of mutant DNA sequences for downstream functional analysis of genetic variants involved in human health and disease. Understanding the mechanisms of different mutagenesis methods can help select the best approach for specific needs. We compared three different approaches for in vivo site-directed DNA mutagenesis that utilize a mutant single-stranded DNA oligonucleotide (ssODN) to target a wild type DNA sequence in the host Escherichia coli (E. coli). The first method, Mandecki, uses restriction nucleases to introduce a double stranded break (DSB) into a DNA sequence which needs to be denatured prior to co-transformation. The second method, recombineering (recombination-mediated genetic engineering), requires lambda red gene products and a mutant ssODN with homology arms of at least 20 nucleotides. In a third method described here for the first time, DNA gap repair, a mutant ssODN targets a DNA sequence containing a gap introduced by PCR. Unlike recombineering, both DNA gap repair and Mandecki can utilize homology arms as short as 10 nucleotides. DNA gap repair requires neither red gene products as recombineering nor DNA denaturation or nucleases as Mandecki, and unlike other methods is background-free. We conclude that Mandecki, recombineering, and DNA gap repair have at least partly different mechanisms, and that DNA gap repair provides a new, straightforward approach for effective site-directed mutagenesis.

scholarly journals Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension

BioTechniques ◽  
2020 ◽  
Vol 68 (6) ◽  
pp. 345-348
Author(s):  
Rasmus Hejlesen ◽  
Ernst-Martin Füchtbauer
Keyword(s):  
Dna Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Overlap Extension

We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair.

scholarly journals A simple method for site-directed mutagenesis using the polymerase chain reaction

1989 ◽  
Vol 17 (21) ◽  
pp. 8915-8915 ◽  
Author(s):  
A. Hemsley ◽  
N. Arnheim ◽  
M.D. Toney ◽  
G. Cortopassi ◽  
D.J. Galas
Keyword(s):  
Polymerase Chain Reaction ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Chain Reaction ◽  
Simple Method ◽  
Polymerase Chain

A novel CRISPR-directed gene editing method that catalyzes precise gene segment replacement in vitro enabling multiplex site-directed mutagenesis.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14688-e14688
Author(s):  
Gabi Tarcic ◽  
Brett M Sansbury ◽  
Amanda M Wagner ◽  
Shaul Barth ◽  
Ester Paniri ◽  
...  
Keyword(s):  
Success Rate ◽  
Plasmid Dna ◽  
Gene Segment ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Simple Method ◽  
Bacterial Colony ◽  
Site Specific ◽  
Ribonucleoprotein Complexes

e14688 Background: Functional analysis of the multitude of mutations found in tumors is a major goal to better understand their role and to optimize patient treatment. PCR-based site-directed mutagenesis (SDM) techniques are often used to engineer these variants. While these tools are efficient, they are not without significant limitations, most notably off-site mutagenesis, limited scalability and lack of multiplexing capabilities. To overcome many of these limitations, we describe a novel, fast and simple method for the introduction of both simple and complex gene mutations in plasmid DNA by using in vitro CRISPR based DNA editing. Methods: For each mutation, a specifically designed pair of CRISPR/Cas12a ribonucleoprotein complexes are used to execute site-specific double-strand breaks on plasmid DNA enabling the excision of a defined DNA fragment. This is followed by donor DNA replacement and bacterial colony expansion. We term this method, CRISPR-directed DNA Mutagenesis (CDM). Results: Using CDM we have been able to synthesize known oncogenic mutations as well as novel variants in 8 different cancer genes. These mutations have been synthesized with over 60% success rate, compared to about 40% success rate in SDM. More importantly, we show that in the CDM method there were no off-site mutations eliminating the need to sequence large portions of the gene. Conclusions: We have developed a novel multiplex site-directed mutagenesis method that can generate multiple unique mutations simultaneously within plasmids. CDM has proven capable of precise, rapid and robust mutation synthesis, including single base point mutations, site-specific deletions, insertions and duplications within targeted plasmids.

A simple method for site-directed mutagenesis with double-stranded plasmid DNA

1998 ◽  
Vol 9 (3) ◽  
pp. 235-241
Author(s):  
Derhsing Lai ◽  
Xuli Zhu ◽  
Sidney Pestka
Keyword(s):  
Plasmid Dna ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Simple Method

scholarly journals Simple and Rapid Assembly of TALE Modules Based on the Degeneracy of the Codons and Trimer Repeats

Genes ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1761
Author(s):  
Lingyin Cheng ◽  
Xiaoqing Zhou ◽  
Yuling Zheng ◽  
Chengcheng Tang ◽  
Yu Liu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Epigenetic Modification ◽  
Cost Effective ◽  
Target Sequence ◽  
Transcription Activator ◽  
Gibson Assembly ◽  
Dna Targeting ◽  
Assembly Method ◽  
Short Palindromic Repeat ◽  
Dna Imaging

Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.

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