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  • BASICS OF MOLECULAR BIOLOGY (II)

    Few areas of molecular biology have not changed with the emergence of a number of techniques subsumed under the generic term for Genetic Engineering and interchangeably referred to as cloning, recombinant DNA or genetic manipulation. Before the development of Genetic Engineering was not possible to isolate a particular eukaryotic gene in sufficient quantities for study molecular or your product.

    ENZYMES:
    There are many enzymes involved in various processes decisively Molecular Biology. These can include:

    Restriction endonucleases: These are enzymes that hydrolyze internucleotide bonds breaking nucleic acids inside the chain. Restriction endonucleases are enzymes produced mainly by bacteria that hydrolyze phosphodiester bonds of the skeleton of double-stranded DNA at specific sequences. Restriction endonucleases of type II are most useful in the methods of DNA due to its absolute sequence specificity for both the binding reaction to the break.

    Restriction enzymes are named with three or four letters that correspond to the first letter of genus and first two or three letters of the species of the source organism. The number indicates the chronological order of discovery of this enzyme in that race.

    Almost all nucleotide sequences recognized by restriction endonucleases possess a symmetry axis of improper binary, that is, reading the sequence in both directions is the same, which is called palindromes. The failure occurs on both strands of DNA being the binary symmetric about the axis.

    Restriction endonucleases cleave DNA to generate either 3 ‘ends or 5 ‘single-stranded, about four nucleotides in length, called cohesive ends or blunt ends.

    synthesize a molecule that is complementary to the template. Polymerases used most often are: DNA polymerase I from E. coli, Klenow fragment, reverse transcriptase (RNA used as template to make DNA), DNA polymerase of bacteriophage T7 RNA polymerase of bacteriophages SP6, T7 and T3 and Taq polymerase (an enzyme used in the chain reaction polymerase). Most polymerases need a little primer or template DNA complementary to the first to initiate the polymerization from that point. The terminal transferase is a polymerase that does not require mold and adds nucleotides only to the ends of existing chains. All require Mg2 +.

    Other Enzymes: Ligases (binds DNA ends protruding or blunt), phosphatases (remove the phosphate from the 5 ‘region of nucleic acids, kinases (incorporated phosphate in the 5’ end left by the phosphatases) etc …

    THE CLONING :
    It consists in obtaining a large number of identical fragments from the original one. This first isolated DNA fragment is to clone, is linked to a carrier or vector (plasmids, phages, cosmids etc. ..) after bringing it into a cell will allow its replication by culture. Once replicated significantly recovered along with the corresponding vector and was subsequently separated from the vector, resulting in a large number of copies of the fragment of interest.

    Electrophoresis :
    A method that allows separation of molecules based on their size. This separation is performed in a gel, a complex mesh of polymer molecules. The gels can be agarose or polyacrylamide. Agarose is suitable for separating nucleic acid fragments in the range from a few hundred, to approximately 20,000 base pairs. The polyacrylamide is preferable for the separation of smaller fragments of nucleic acids.

    The rationale behind this separation is based on the nucleic acid molecules are negatively charged (pH and buffer conditions in which they are) and when subjected to an electric field migrate toward the positive pole of this through the gel, to speeds depending on their sizes: a small molecule can continue its path through the gel more easily than a large molecule. The migration velocities of the molecules of nucleic acids, are inversely proportional to the logarithm of their molecular weights (Aaij and Borst). Detection after migration is easily accomplished through the use of an intercalating dye (intercalates between nucleic acid bases) containing the gel itself and is displayed by emitting visible light when the gel is illuminated with ultraviolet light. One factor to take into account in assessing the migration of nucleic acids in a gel as well as its size is the spatial configuration to take the molecule.

    Hybridization :
    It is a process of joining two complementary strands of nucleic acid but whose origin is different. One will be the target nucleic acid and the other is the probe used to locate the target nucleic acid.

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