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    4 Basic Steps of Genetic Recombination Engineering

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    shainemary niones
    The 4 basic steps of genetic recombination engineering are: 1) isolating the target gene and vector, 2) digesting the gene and vector with restriction enzymes to generate complementary ends, 3) ligating the gene insert into the vector using DNA ligase to form a recombinant construct, and 4) selecting and expressing the recombinant construct by introducing it into a host cell where the gene of interest can be expressed.

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    4 Basic Steps of Genetic Recombination Engineering

    Uploaded by

    shainemary niones
    27 views1 page
    The 4 basic steps of genetic recombination engineering are: 1) isolating the target gene and vector, 2) digesting the gene and vector with restriction enzymes to generate complementary ends, 3) ligating the gene insert into the vector using DNA ligase to form a recombinant construct, and 4) selecting and expressing the recombinant construct by introducing it into a host cell where the gene of interest can be expressed.

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    4 BASIC STEPS OF GENETIC RECOMBINATION ENGINEERING

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    The 4 basic steps of genetic recombination engineering are: 1) isolating the target gene and vector, 2) digesting the gene and vector with restriction enzymes to generate complementary ends, 3) ligating the gene insert into the vector using DNA ligase to form a recombinant construct, and 4) selecting and expressing the recombinant construct by introducing it into a host cell where the gene of interest can be expressed.

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    Download as docx, pdf, or txt
    27 views1 page

    4 Basic Steps of Genetic Recombination Engineering

    Uploaded by

    shainemary niones
    The 4 basic steps of genetic recombination engineering are: 1) isolating the target gene and vector, 2) digesting the gene and vector with restriction enzymes to generate complementary ends, 3) ligating the gene insert into the vector using DNA ligase to form a recombinant construct, and 4) selecting and expressing the recombinant construct by introducing it into a host cell where the gene of interest can be expressed.

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    4 BASIC STEPS OF GENETIC RECOMBINATION ENGINEERING

    1. ISOLATING GENE AND VECTOR


     The target DNA or gene to be cloned must be first isolated. A gene of interest is a
    fragment of gene whose product (a protein, enzyme or a hormone) interests us. For
    example, gene encoding for the hormone insulin. The gene of interest can then be
    specifically amplified via the polymerase chain reaction (PCR)
     A vector is a DNA molecule that is used as a vehicle to carry the gene of interest into a
    foreign cell. Bacterial plasmids are commonly used as vectors because they are capable
    of autonomous self-replication and expression.

    2. DIGESTION WITH RESTRICTION ENZYMES


     In order to incorporate a gene of interest into a vector, both must be cut with
    restriction enzymes at specific recognition sites.
     Different restriction enzymes recognize and cut different DNA sequences at specific
    nucleotide sequences.
     Restriction enzymes cleave the sugar-phosphate backbone to generate blunt
    ends or sticky ends (complementary overhangs).
    3. LIGATION OF VECTOR AND INSERT
     The gene of interest is inserted into a plasmid vector that has been cut with the same
    restriction endonucleases
     This occurs because the sticky ends of the gene and vector overlap via complementary
    base pairing
     The gene and vector are then spliced together by the enzyme DNA ligase to form
    a recombinant construct
     DNA ligase joins the vector and gene by fusing their sugar-phosphate backbones
    together.
    4. SELECTION AND EXPRESSION
     The recombinant construct (including the gene of interest) is finally introduced into an
    appropriate host cell or organism
     This process can be achieved in a variety of ways and is called transfection (for
    eukaryotes) or transformation (for prokaryotes)
     Antibiotic selection is commonly used in order to identify which cells have successfully
    incorporated the recombinant construct
     The plasmid vector contains an antibiotic resistance gene, so only transgenic cells will
    grow in the presence of antibiotic
     Transgenic cells, once isolated and purified, will hopefully begin expressing the desired
    trait encoded by the gene of interest

    RESTRICTION ENZYMES

     These are proteins isolated from bacteria used in cutting DNA molecules. 
     Specific restriction enzymes cut specific DNA molecule.
     Using for cutting desired genes and vector

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