A rapid PCR dependent microtitre plate screening method for DNA sequences altered by site-directed mutagenesis

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.

Download Full-text

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

10.1101/313155 ◽

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.

Download Full-text

Site-Directed Mutagenesis Using a Rapid PCR-Based Method

In Vitro Mutagenesis Protocols ◽

10.1385/0-89603-332-5:239 ◽

2003 ◽

pp. 239-248 ◽

Cited By ~ 3

Author(s):

Gina L. Costa ◽

John C. Bauer ◽

Barbara McGowan ◽

Mila Angert ◽

Michael P. Weiner

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Rapid Pcr

Download Full-text

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

BioTechniques ◽

10.2144/btn-2019-0104 ◽

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.

Download Full-text

A Novel Simple and Rapid PCR-Based Site-Directed Mutagenesis Method

Molecular Biotechnology ◽

10.1385/mb:26:1:27 ◽

2004 ◽

Vol 26 (1) ◽

pp. 27-34 ◽

Cited By ~ 22

Author(s):

Imen Rabhi ◽

Naouel Guedel ◽

Imen Chouk ◽

Khaled Zerria ◽

M. Ridha Barbouche ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Rapid Pcr

Download Full-text

Different simple protocols for separating different types of coexisted plasmids in the same cell

Biotechnology and Bioprocessing ◽

10.31579/2766-2314/027 ◽

2021 ◽

Vol 2 (3) ◽

pp. 01-06

Author(s):

Amro Amara

Keyword(s):

Single Gene ◽

Screening Method ◽

The Other ◽

Site Directed Mutagenesis ◽

Natural Phenomenon ◽

Directed Mutagenesis ◽

Experimental Conditions ◽

E Coli ◽

Competent Cells ◽

Transformation Processes

The existence of mixed plasmids in the same cell is tricky and there is a need for separating them from each other. However, isolating two existed plasmids might be difficult, particularly if they are same in their sizes, with same antibiotic marker, or different only in one or more mutants without different restriction cut. Two different plasmids in the same cells is a natural phenomenon as well as a normal practice in molecular biology experiments. For example during random mutagenesis experiment for a single gene (existed naturally in an operon) using a mutator strain like E. coli XL1 Red, the single mutated gene is then complemented with the other essential genes for producing certain products. Another example, during site directed mutagenesis experiment using double antibiotics selection method, in many cases, the original plasmid is existed side by side with the one carry the new mutant. There are many examples where plasmids coexisted with each other either naturally or under experimental conditions. The problem is how one could separate those plasmids particularly when they are similar in their molecular weight and have the same marker. This study introduces two main strategies; the first is based on increasing or decreasing the competent cells transformation efficacy. Where, in general harvesting competent cells either E. coli or other bacterial strains in the first 2-3 hours (or less) of their cultivation and using the enhanced protocol for competent cells preparation will improve the transformation processes. Letting cells to be more ages will reduce the transformation processes. Using four 2-3 hour grown competent Azotobacter sp enable plasmid transformation. The second strategy for separating the coexisted plasmid is based on using diluted plasmids. The antibiotic screening method is based on blind selection where growing on plat containing the first antibiotic and non growing in the second antibiotic means that the tow existed plasmids are separated. In case of existing of plasmids with the same size and the same antibiotic marker for example during the site-directed-mutagenesis protocol (mutants did not have different restriction enzymes cut), the plasmid is diluted and transformed in recombinant E. coli and each clone was cultivated alone and the mutated region is sequenced. The presence of a single base pair in the site of the mutant means presence of a single plasmids and vice versa. As a conclusion same plasmids with point mutation are usually coexisted. In some cases the coexisted plasmids are with similar antibiotic marker, no different restriction enzyme cut sites are existed, no white and blue selection or any other phenotype for selection. In such cases and similar ones diluting plasmid and transforming them in conditions enable single plasmid per cell must be controlled by the sequencer. The protocols included in this study are summarized from the experiences with random and site directed mutagenesis experiments where plasmid with a single mutant is coexisted with the wild mother plasmid or with the other coexisted different mutants.

Download Full-text

Rapid PCR site-directed mutagenesis.

Genome Research ◽

10.1101/gr.4.3.s131 ◽

1994 ◽

Vol 4 (3) ◽

pp. S131-S136 ◽

Cited By ~ 39

Author(s):

M P Weiner ◽

G L Costa

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Rapid Pcr

Download Full-text

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

10.21203/rs.3.rs-1123734/v1 ◽

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%).

Download Full-text