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Molekuliarnaia Biologiia 2016"Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful... (Review)
Review
"Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.
Topics: Cloning, Molecular; DNA, Recombinant; Genetic Engineering; Genetic Vectors
PubMed: 27028806
DOI: 10.7868/S0026898416010134 -
Nucleic Acids Research Nov 1979A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones...
A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.
Topics: Animals; Chromosomes, Bacterial; Cloning, Molecular; DNA Restriction Enzymes; DNA, Circular; DNA, Recombinant; Escherichia coli; Methods; Mice; Molecular Weight; Plasmids; Transformation, Bacterial
PubMed: 388356
DOI: 10.1093/nar/7.6.1513 -
Methods in Molecular Biology (Clifton,... 2017QuickStep-Cloning is a novel molecular cloning technique that builds upon the concepts of asymmetric PCR and megaprimer-based amplification of whole plasmid. It was...
QuickStep-Cloning is a novel molecular cloning technique that builds upon the concepts of asymmetric PCR and megaprimer-based amplification of whole plasmid. It was designed specifically to address the major drawbacks of previously reported cloning methods. The fully optimized protocol allows for a seamless integration of a long DNA fragment into any position within a plasmid of choice, in a time-efficient and cost-effective manner, without the need of a tedious DNA gel purification, a restriction digestion, and an enzymatic ligation. QuickStep-Cloning can be completed in less than 6 h, significantly faster than most of the existing cloning methods, while retaining high efficiency.
Topics: Cloning, Molecular; DNA, Recombinant; Plasmids; Polymerase Chain Reaction; Recombination, Genetic; Transformation, Bacterial
PubMed: 27671943
DOI: 10.1007/978-1-4939-6343-0_16 -
Pharmacological Reports : PR 2013Pharmaceutical biotechnology has a long tradition and is rooted in the last century, first exemplified by penicillin and streptomycin as low molecular weight... (Review)
Review
Pharmaceutical biotechnology has a long tradition and is rooted in the last century, first exemplified by penicillin and streptomycin as low molecular weight biosynthetic compounds. Today, pharmaceutical biotechnology still has its fundamentals in fermentation and bioprocessing, but the paradigmatic change affected by biotechnology and pharmaceutical sciences has led to an updated definition. The biotechnology revolution redrew the research, development, production and even marketing processes of drugs. Powerful new instruments and biotechnology related scientific disciplines (genomics, proteomics) make it possible to examine and exploit the behavior of proteins and molecules. Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. This technology, now approximately 25 years old, is becoming one of the most important technologies developed in the 20(th) century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances regarding rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we describe these processes.
Topics: Animals; Biotechnology; DNA, Recombinant; Darbepoetin alfa; Erythropoietin; Fibrinolytic Agents; Genetic Engineering; Genomics; Hematinics; Humans; Hypoglycemic Agents; Insulin, Regular, Human; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Proteomics; Recombinant Proteins; Somatostatin; Tissue Plasminogen Activator
PubMed: 24399704
DOI: 10.1016/s1734-1140(13)71466-x -
Journal of Virology Aug 1996We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helper-free retrovirus with titers of 1 X 10(6) to 5 X 10(6)...
We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helper-free retrovirus with titers of 1 X 10(6) to 5 X 10(6) within 48 h. These vectors contain the immediate early region of the human cytomegalovirus enhancer-promoter fused to the Moloney murine leukemia virus long terminal repeat at the TATA box in the 5' U3 region, yielding the pCL promoter. By selecting vectors designed to express genes from one of four promoters (dihydrofolate reductase, Rous sarcoma virus, long terminal repeat, or cytomegalovirus), the pCL system permits the investigator to control the level of gene expression in target cells over a 100-fold range, while maintaining uniformly high titers of virus from transiently transfected producer cells. The pCL packaging plasmids lack a packaging signal (delta-psi) and include an added safety modification that renders them self-inactivating through the deletion of the 3' U3 enhancer. Ecotropic, amphotropic (4070A), and amphotropic-mink cell focus-forming hybrid (10A1) envelope constructions have been prepared and tested, permitting flexible selection of vector pseudotype in accordance with experimental needs. Vector supernatants are free of helper virus and are of sufficiently high titer within 2 days of transient transfection in 293 cells to permit infection of more than 50% of randomly cycling target cells in culture. We demonstrated the efficacy of these vectors by using them to transfer three potent cell cycle control genes (the p16(INK4A), p53, and Rb1 genes) into human glioblastoma cells.
Topics: DNA, Recombinant; DNA, Viral; Genetic Vectors; Humans; Retroviridae
PubMed: 8764092
DOI: 10.1128/JVI.70.8.5701-5705.1996 -
Cell Jun 2016Denvaxia is the first licensed vaccine for the prevention of dengue. It is a live vaccine developed using recombinant DNA technology. The vaccine is given as three doses...
Denvaxia is the first licensed vaccine for the prevention of dengue. It is a live vaccine developed using recombinant DNA technology. The vaccine is given as three doses over the course of a year and has the potential to prevent hundreds of thousands of hospitalizations each year.
Topics: DNA, Recombinant; Dengue; Dengue Vaccines; Humans
PubMed: 27368091
DOI: 10.1016/j.cell.2016.06.036 -
Annals of Surgery Aug 1990We have the ability to isolate DNA from tissue, determine its base-pair sequence, and ask if a gene of interest is present. DNA strands can be isolated from one type of... (Review)
Review
We have the ability to isolate DNA from tissue, determine its base-pair sequence, and ask if a gene of interest is present. DNA strands can be isolated from one type of cell or organism, cleaved, and inserted (recombined) with DNA from another cell or organism. Recombinant DNA techniques have already improved health care by providing clinically useful quantities of pure human protein hormones such as erythropoietin, insulin, and growth hormone. Furthermore these techniques may increase our understanding of cellular growth control mechanisms to a level that was previously unattainable. They will also increase our knowledge of the development of major diseases and provide a means of specific nontoxic therapies for these diseases. Surgeons will need to understand basic DNA research terminology to keep up with the revolution in medical therapies that these techniques will cause. Our purpose is to begin the process of linking surgery to DNA.
Topics: DNA Probes; DNA, Recombinant; General Surgery; Genetic Engineering; Genetic Therapy; Humans; Immunoblotting; Oncogenes; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length
PubMed: 1973891
DOI: 10.1097/00000658-199008000-00011 -
Molecular and Cellular Biology Mar 2014Fred Sherman was a prominent yeast geneticist and my mentor in graduate school. Fred passed away in September 2013 at the age of 81. In this minireview, I describe what... (Review)
Review
Fred Sherman was a prominent yeast geneticist and my mentor in graduate school. Fred passed away in September 2013 at the age of 81. In this minireview, I describe what it was like to know Fred and be in his lab from 1977 to 1982, the extraordinarily exciting time when the recombinant DNA revolution hit yeast genetics.
Topics: DNA, Recombinant; Genetics; Yeasts
PubMed: 24379440
DOI: 10.1128/MCB.01682-13 -
Current Issues in Molecular Biology Jan 2000This article describes the sequence-specific isolation and purification of intact double-stranded DNA (dsDNA) by oligonucleotide/PNA-assisted affinity capture (OPAC).... (Review)
Review
This article describes the sequence-specific isolation and purification of intact double-stranded DNA (dsDNA) by oligonucleotide/PNA-assisted affinity capture (OPAC). The OPAC assay is based on selective tagging of a DNA duplex by biotinylated oligodeoxyribonucleotide (ODN) through formation of a so-called PD-loop. The PD-loop is assembled with the aid of a pair of PNA "openers", which allow sequence-specific targeting with a Watson-Crick complementary ODN probe in the exposed region of the dsDNA. The protocol involves three steps. First, two cationic bis-PNAs locally pry the DNA duplex apart at a predetermined site. Then, the exposed DNA single strand is targeted by a complementary biotinylated ODN to selectively form a stable PD-loop complex. Finally, the capture of dsDNA is performed using streptavidin covered magnetic beads. The OPAC procedure has many advantages in the isolation of highly purified native DNA over other affinity capture and amplification techniques.
Topics: Base Pairing; Biotinylation; DNA; DNA, Fungal; DNA, Recombinant; DNA, Single-Stranded; Magnetics; Microspheres; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Saccharomyces cerevisiae; Streptavidin
PubMed: 11464918
DOI: No ID Found -
Microbial Biotechnology Nov 2013Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are... (Review)
Review
Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.
Topics: Animals; Arthropod Proteins; Biopolymers; Biotechnology; DNA, Recombinant; Recombinant Proteins; Silk
PubMed: 24119078
DOI: 10.1111/1751-7915.12081