A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study

2005 ◽  
Vol 288 (6) ◽  
pp. C1273-C1278 ◽  
Author(s):  
Jae-Kyun Ko ◽  
Jianjie Ma
Keyword(s):  
Molecular Biology ◽  
Protein Engineering ◽  
Restriction Enzyme ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Recognition Site ◽  
Cell Physiology ◽  
Multiple Site ◽  
Functional Studies ◽  
Engineering Studies

PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology.

scholarly journals KARAKTERISASI FRAGMEN DNA GEN GLUKOAMILASE (GLU1) PRODUK PCR DENGAN ANALISIS RESTRIKSI

2005 ◽  
Vol 11 (1) ◽  
pp. 81-79
Author(s):  
Sofijan Hadi
Keyword(s):  
Nucleotide Sequence ◽  
Dna Sequencing ◽  
Restriction Enzyme ◽  
Restriction Enzymes ◽  
Positive Control ◽  
Recognition Site ◽  
Base Pairs ◽  
Saccharomycopsis Fibuligera ◽  
Glucoamylase Gene ◽  
Eco Ri

Characterization used retriction enzyme on the 1784 bp (base pairs) DNA fragmen of glucoamylase gene (GLUI) of E. fibuligera ITB. R. cc. 64 has been done. The rectriction enzyme usage was Stu 1, Eco RI, Eco RV, Bam HI and Sau 3A. The purpose of this research were: First was to know recognition site of 1784 bp DNA fragmen of glucoamylase gene (GLUI) by the restriction enzyme above. The second was to know homologyst the glucoamylase gene (GLUI) E. fibuligera ITB. R. cc. 64 and the glucoamylase gene (GLUI) Saccharomycopsis fibuligera HUT 7212 (pSf GLUI). The result of amplification glucoamylase gene (GLUI) indicated that 1784 bp DNA fragmen on GLUI locus has succesfully isolated and gave the same size with the positive control pSf GLUI. Analysis of those DNA fragmen by StuI, Eco RV, Eco RI, Bam HI and Sau 3A indicated that 1784 bp of DNA fragmen from E. fibuligera ITB.R.cc.64. has the same result with 1784 bp of DNA fragmen from pSf GLUI. The result of the fragments after incubated by restriction enzymes are as follows: 997 bp and 787 bp by Eco RI, 1000 bp and 1780 bp by Bam H) and 850 bp and 760 bp by Sau 3A. Digestion using StuI and Eco RI was failed. To ensure that the DNA fragmen 1784 bp has characteristic as glucoamylase gene, it should be expressed into S. cerevisiae and/or should be determined the nucleotide sequence by DNA sequencing.

scholarly journals The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Anurag Kumar Sinha ◽  
Kristoffer Skovbo Winther
Keyword(s):  
Active Sites ◽  
Molecular Switch ◽  
Cell Physiology ◽  
Synthetase Activity ◽  
Wild Type ◽  
Functional Studies ◽  
Loop Region ◽  
A Site ◽  
Trna Binding

AbstractBacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity remain elusive. Here, we show a role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using mutagenesis screening and functional studies, we identify a loop region (residues 114–130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wild type RelA. Our data support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

scholarly journals Membrane Protein Stabilization Strategies for Structural and Functional Studies

Membranes ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 155
Author(s):  
Ekaitz Errasti-Murugarren ◽  
Paola Bartoccioni ◽  
Manuel Palacín
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Membrane ◽  
Protein Stabilization ◽  
New Drugs ◽  
Cell Physiology ◽  
Protein Preparation ◽  
Functional Studies ◽  
Membrane Protein Stability ◽  
Druggable Targets

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

scholarly journals Genome-Wide Identification of 5-Methylcytosine Sites in Bacterial Genomes By High-Throughput Sequencing of MspJI Restriction Fragments

2021 ◽  
Author(s):  
Brian P. Anton ◽  
Alexey Fomenkov ◽  
Victoria Wu ◽  
Richard J. Roberts
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Specific Sequence ◽  
Genome Wide ◽  
Cost Effective Alternative ◽  
Simple Column ◽  
Sequencing Platforms

ABSTRACTSingle-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

scholarly journals Computation of Splicing Languages from DNA Splicing System Based on Sequences of Restriction Enzymes

2019 ◽  
Vol 8 (6S3) ◽  
pp. 31-42
Keyword(s):  
Restriction Enzyme ◽  
Dna Computing ◽  
Restriction Enzymes ◽  
Visual Programming ◽  
Cleavage Pattern ◽  
Dna Molecules ◽  
Splicing Systems ◽  
Dna Splicing

In DNA splicing systems, restriction enzymes and ligases cleave and recombine DNA molecules based on the cleavage pattern of the restriction enzymes. The set of molecules resulting from the splicing system depicts a splicing language. In this research, an algorithm for DNA splicing systems is developed using C++ visual programming. The splicing languages which have been characterised through some theorems based on the crossings and sequences of the restriction enzymes, are generated as the output from this computation. In order to generate the splicing languages, the algorithm detects and calculates the number of cutting sites of the restriction enzymes found in the initial molecules, determines whether the sequence of restriction enzyme is a palindrome or not, and if the restriction enzymes have the same or different crossings. The results from this research depict the splicing languages obtained from the manual computations, which contributes to the development of computational software in DNA computing.

scholarly journals Engineering a Minimal Cloning Vector from a pUC18 Plasmid Backbone with an Extended Multiple Cloning Site

2018 ◽  
Author(s):  
Jens Staal ◽  
Wouter De Schamphelaire ◽  
Rudi Beyaert
Keyword(s):  
Synthetic Biology ◽  
Molecular Biology ◽  
Restriction Enzyme ◽  
Essential Role ◽  
Building Blocks ◽  
Cloning Vector ◽  
Multiple Cloning Site ◽  
Unique Restriction ◽  
Plasmid Backbone

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.

scholarly journals Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0247541
Author(s):  
Brian P. Anton ◽  
Alexey Fomenkov ◽  
Victoria Wu ◽  
Richard J. Roberts
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Specific Sequence ◽  
Genome Wide ◽  
Cost Effective Alternative ◽  
Simple Column ◽  
Sequencing Platforms

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

scholarly journals Out-Lab Therapy Approach Based on Elected A Restriction Enzyme to Transfer Target Gene

2018 ◽  
Vol 2 (2) ◽  
pp. 196-208
Author(s):  
Ayad Ismaeel
Keyword(s):  
Restriction Enzyme ◽  
Target Gene ◽  
Restriction Enzymes ◽  
Tp53 Gene ◽  
Restriction Enzyme Digestion ◽  
Therapy Approach ◽  
Software Packages ◽  
Important Approach ◽  
Effective Cost ◽  
Target Gene Sequence

An important approach of therapy the target gene sequence causes diseases via repair/recombine the mutated gene (gene transfer) using a restriction enzymes in the laboratory. This approach will cause multiple problems happening accompany to biological laboratory if ruled out problems outside of it like the digested DNA ran as a smear on an agarose gel, incomplete restriction enzyme digestion, extra bands in the gel, etc. The paper suggested new approach of therapy via repair/replacement mutated gene caused disease by detecting primers and finding restriction enzymes using bioinformatics tools, software, packages etc. then achieving the repair/ recombine of mutations before going to the biologic lab (out-lab) to avoid the problems associated these laboratories. Implement and apply this a proposed therapy approach on TP53 gene (which caused more than 50% of human cancers) and after confirming there is mutations on P53 tumor protein shows an effective cost, friendly therapy methodology and comprehensive.

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