Artificial Restriction Enzymes As Tools for Future Molecular Biology and Biotechnology

2006 ◽  
pp. 159-175
Author(s):  
Yoji Yamamoto ◽  
Makoto Komiyama
Keyword(s):  
Molecular Biology ◽  
Restriction Enzymes ◽  
Artificial Restriction

Programmable DNA-Guided Artificial Restriction Enzymes

2017 ◽  
Vol 6 (5) ◽  
pp. 752-757 ◽  
Author(s):  
Behnam Enghiad ◽  
Huimin Zhao
Keyword(s):  
Restriction Enzymes ◽  
Artificial Restriction

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 Straightforward Procedure for Laboratory Production of DNA Ladder

2012 ◽  
Vol 2012 ◽  
pp. 1-4 ◽  
Author(s):  
Vo Thi Thuong Lan ◽  
Pham Thi Thanh Loan ◽  
Pham Anh Thuy Duong ◽  
Le Thi Thanh ◽  
Ngo Thi Ha ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Tandem Repeat ◽  
Restriction Site ◽  
Restriction Enzymes ◽  
Dna Fragments ◽  
Repeated Dna ◽  
Dna Ladder ◽  
Time Saving ◽  
Partial Digestion ◽  
Unique Restriction

DNA ladder is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare a DNA ladder that consists of 10 fragments from 100 to 1000 bp. This protocol is a combination of routinely employed methods: cloning, PCR, and partial digestion with restriction enzymes. DNA fragments of 100 bp with unique restriction site at both ends were self-ligated to create a tandem repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments. Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.

scholarly journals Mining for Restriction Endonucleases in Nicaragua

Encuentro ◽  
2012 ◽  
pp. 49-62
Author(s):  
Suyén E. Espinoza-Miranda ◽  
Julio A. Gómez-Rodríguez ◽  
Jorge Huete-Pérez
Keyword(s):  
Molecular Biology ◽  
Central America ◽  
Restriction Enzymes ◽  
Research Capacity ◽  
Central American ◽  
Industrial Enzymes ◽  
Biotechnology Research ◽  
Rdna Sequencing ◽  
Modern Molecular Biology ◽  
The University

The Molecular Biology Center at the University of Central America in Nicaragua(CBM-UCA) was founded in 1999 to strengthen biotechnology research capacity and education in Nicaragua and the Central American region. One of the first projects launched by the CBM-UCA was bio-prospecting for key industrial enzymes. This ongoing study seeks to discover and characterize restriction enzymes (RE) in bacteria, and to create a database of microorganisms isolated and identified by 16S rDNA sequencing methodology. In this paper we highlight the importance of studying the extreme environmental conditions for building knowledge of Nicaraguan biodiversity through modern molecular biology techniques such as metagenomics. The isolation of prototype enzymes such as EcoRV and ClaI is presented as an update and extension of previously undertaken work.

scholarly journals How the techniques of molecular biology are developed from natural systems

2018 ◽  
Author(s):  
Isobel Ronai
Keyword(s):  
Nucleic Acid ◽  
Molecular Biology ◽  
Molecular Mechanisms ◽  
Fluorescent Proteins ◽  
Restriction Enzymes ◽  
Developmental Trajectory ◽  
Molecular Biology Technique ◽  
Natural Systems ◽  
Biological Specificity ◽  
Specificity Protein

A striking characteristic of the highly successful techniques in molecular biology is that they are derived from natural occurring systems. RNA interference (RNAi), for example, utilises a mechanism that evolved in eukaryotes to destroy foreign nucleic acid. Other examples include restriction enzymes, the polymerase chain reaction, fluorescent proteins and CRISPR-Cas9. I propose that natural molecular mechanisms are exploited by biologists for their effectors’ (protein or nucleic acid) activity and biological specificity (protein or nucleic acid can cause precise reactions). I also show that the developmental trajectory of novel techniques in molecular biology, such as RNAi, is four characteristic phases. The first phase is discovery of a biological phenomenon. The second is identification of the mechanism’s trigger(s), the effector and biological specificity. The third is the application of the technique. The final phase is the maturation and refinement of the molecular biology technique. The development of new molecular biology techniques from nature is crucial for both biological and biomedical research.

scholarly journals Penyisipan Gen Inhibitor α-amilase pada Plasmid Biner pCambia 1301

2016 ◽  
Vol 1 (2) ◽  
pp. 45
Author(s):  
Edy Listanto ◽  
Sutrisno Sutrisno ◽  
Saptowo J. Pardal ◽  
M. Herman
Keyword(s):  
Molecular Biology ◽  
Research And Development ◽  
Genetic Resources ◽  
Restriction Enzymes ◽  
Restriction Pattern ◽  
E Coli ◽  
Colony Number ◽  
The Right ◽  
Biology Laboratory ◽  
Binary Plasmid

<p class="p1">The experiment was conducted at the Molecular Biology Laboratory of the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor. The objective was to construct <em>-ai </em>gene on a binary plasmid <em>p</em>Cambia 1301. This experiment was carried out using construction method by ligation process between fragments of <em>α-ai </em>gene from <em>p</em>TA<span class="s1">3 </span>plasmid and <em>p</em>Cambia 1301 on <em>Hind</em>III site. The result of ligant transformation into <em>E. coli </em>DH5<em>α </em>was 182 surviving colonies on YEP medium containing kanamycin. DNA samples were obtained from 60 randomly selected colonies. The restriction pattern was tested by digesting each DNA sample using <em>Hind</em>III showed colonies containing two fragments expected of sizes wich are 11.837 and 4.887 kb. Two colonies are predicted containing of <em>α-ai </em>gene on its the binary plasmid. Advanced tests using restriction enzymes <em>Bam</em>HI and <em>Xba</em>I showed two directions (right and left) of <em>α-ai </em>gene. The right direction was shown by <em>p</em>Cambia-<em>α-ai</em>1 from colony number 43. This plasmid showed expected fragments of sizes 13.485 and 3.219 kb when digested with <em>Bam</em>HI and two fragments of sizes 15.421 and 1.303 kb when digested with <em>Xba</em>I. The left direction was shown <em>p</em>Cambia-<em>α-ai</em>2 from colony number 58. This plasmid also demon-strated expected fragments of sizes 15.026 and 1.698 kb when digested with <em>Bam</em>HI and two fragments of sizes 13.082 and 3.642 kb when digested with <em>Xba</em>I. Both <em>p</em>Cambia-<em>α-ai</em>1 and <em>p</em>Cambia-<em>α-ai</em>2 were transformed into <em>A. tumefaciens </em>LBA4404.</p>

scholarly journals Restriction enzymes and their use in molecular biology: An overview

2019 ◽  
Vol 44 (2) ◽  
Author(s):  
Francesca Di Felice ◽  
Gioacchino Micheli ◽  
Giorgio Camilloni
Keyword(s):  
Molecular Biology ◽  
Restriction Enzymes
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