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    Displaying items by tag: DNA cloning
    Tuesday, 15 April 2014 09:52

    Gene editing cures rare liver disease

    CRISPR is very easy to configure and customize equipmentUsing a new system of genetic editing based on bacterial proteins by researchers from MIT cured rare liver disease caused by a single genetic mutation.

    The findings described in the edition of Nature Biotechnology, provide the first evidence that the technique of editing of a gene known as CRISPR, can reverse disease symptoms.

    CRISPR, which offers an easy way to crop the mutated DNA and replacement with the correct sequence has the potential to treat many genetic diseases, according to the research team.

    Recently developed CRISPR system relies on cellular mechanism that bacteria use to protect themselves from viral infection.

    Researchers have copied this cell system for the creation of gene-editing complexes, including DNA.

    They are cut enzyme called Cas9, bound to the RNA strand, which can be programmed to bind to a specific genomic sequence.

    Meanwhile, researchers deliver DNA template strand.

    When repairing cell damage resulting from Cas9, scientists introduced the template DNA in the genome.

    Scientists predict that this type of revision of the genome one day could help in the treatment of diseases such as hemophilia, Huntington's disease, and the like, caused by a single mutation.

    There are other systems developed on the basis of genetic editing of DNA enzymes known as nucleases, but these complexes can be expensive and difficult to assemble.

    In contrast, CRISPR is very easy to configure and customize equipment.

    Published in News

    13-researchersc-gene-targeting-rosa26-feeder-cells-human-primary-cells-mef cells-rat

    A computational model developed by researchers at Rensselaer Polytechnic Institute is the first to accurately simulate the complex twists of a short sequence of RNA as it folds into a critical hairpin structure known as a "tetraloop." The research, published today in Proceedings of the National Academy of Sciences, is a glimpse into RNA, found in all life on Earth, and could advance a variety of research areas, including the search for new antibiotics and cures for protein-related diseases.
    Existing computational models, based on DNA rather than RNA, do not achieve the atomic level accuracy of the new model, said Angel Garcia, head of the Department of Physics, Applied Physics, and Astronomy within the School of Science at Rensselaer, and senior constellation chaired professor in the Biocomputation and Bioinformatics Constellation, who co-wrote the paper with Alan Chen, a post-doctoral fellow at Rensselaer. The new model Garcia and Chen created can simulate the folding of three known versions of a tetraloop, accurate to within one ten-billionth of a meter.
    RNA is involved in many biological functions, such as building proteins, coding and decoding genes, and cellular regulation. RNA molecules are composed of strings of four different "bases" —cytosine, guanine, adenine, and uracil—mounted on a sugar-phosphate backbone. Once the sequence is assembled, the individual bases interact with their neighbors, twisting and swinging on the hinged chemical bonds that connect them to the backbone. When the process is complete, the RNA has folded into its "tertiary" structure, which influences its function. Although researchers can easily alter the sequence of molecules, without accurate computer modeling there they cannot easily see the tertiary structure of their creation.
    "Right now, it takes people from molecular biologists, to virologists, to cell biologists, thousands of dollars and years of study to see the structure of an RNA they have made, altered, or are studying," said Chen. "There are a lot of researchers working on the RNA in viruses and how it attacks the cell, and, while they're easily able to alter the sequence, they're essentially working without ever seeing the effects of their changes in molecular detail. Because of this, there's a lot of trial and error, and our work aimed at helping that."
    Garcia and Chen said that, unlike DNA, which typically twists two strands of bases into a classic double-helix, RNA is single-stranded and folds onto itself, forming many unusual structures. A tetraloop is a small section of single-stranded RNA that is looped into the shape of a hairpin, the curve of which is formed by four bases. Even the sequence of bases in a tetraloop is unusual, violating a standard arrangement described by groundbreaking DNA researchers James Watson and Francis Crick.
    To create an effective computational model, Garcia and Chen had to match the unique "recipe" of twisting and swinging proscribed by the interactions between the bases.
    "Imagine if you try to produce a recipe of Mario Batali," said Garcia, referring to a popular chef. "I tell you it has water, salt, fish, and pasta—go produce his recipe. The problem is, you don't know how much of each, and in what order."
    Instead of a recipe of food ingredients, Garcia and Chen created a computational recipe for the interactions of the bases in the sequence of a tetraloop.
    "The problem is one of balancing different forces. It's the actions between the bases as they stack on top of each other, the interactions of the bases with water, the rotation of the bases relative to a sugar. Those are things that change the balance," said Garcia.
    Garcia said tetraloops are an important area of study because they appear in all organisms, particularly in ribosomes, which manufacture proteins for living cells. Statistically, there could be as many as 256 possible sequences of those four bases, but only three sequences actually appear in tetraloops. Once formed, they are highly stable, outlasting other structures when subjected to the destructive force of increasing heat.
    "Tetraloops are sequences which are highly conserved throughout evolution; you find them everywhere, from bacteria to humans," said Garcia. "From one organism to another, many things can change, but when tetraloops change, they change from one sequence of four bases to one of the other three. They stack against each other and they are hyperstable. And there is a reason for them to be arranged the way they are."

    Published in News
    Monday, 11 March 2013 15:49

    DNA Cloning Service

    With decades of experience our Molecular Biology Team has developed an exceptional expertise in anything DNA-related. We  can help you develop the best strategy to tackle your cloning projects and find solutions to all your technical problems. 

    Some examples of our services:

    • - Cloning of amplified DNA fragments
    • - Gene targeting vectors for homologous recombination
    • - Bacterial Artificial Chromosome (BAC) Recombineering
    • - RNAi and inducible vectors
    • - Site-specific mutagenesis
    • - Tagging of genes
    • - De novo gene synthesis

     

    A typical cloning procedure includes:

    • - Primer design for target region
    • - PCR amplification of target region
    • - Cloning of the PCR product into a cloning vector
    • - Sub-cloning the gene into the vector of your choice
    • - Picking, plating, culturing, and DNA preparation
    • - Sequence confirmation
    • - Plasmid purification (Mini, Midi, or Maxi size)
    Published in Targatt services