genetic engineering

methods to isolate desired gene (2)

-use restriction endonucleases

-use reverse transcriptase

isolating gene using restriction endonucleases

-desired gene is isolated using restriction endonucleases

-the restriction endonucleases cuts DNA at specific recognition sequences, producing sticky ends

isolating gene using reverse transcriptase

-mRNA is extracted from cell that produces desired protein

-Reverse transcriptase converts mRNA into complementary DNA (cDNA)

recombinant DNA

DNA formed by combining DNA from 2 different sources

transgenic organism/ genetically modified organism

an organism that contains a gene from another species in its genome

e.g. Bacteria with human insulin gene → produce insulin

Formation of Recombinant DNA process

-desired gene is isolated using restriction endonucleases

-the restriction endonucleases cuts DNA at specific recognition sequences, producing sticky ends

-a plasmid vector is cut using the same restriction enzyme, creating complementary sticky ends.

-the complementary sticky ends anneal

-DNA ligase joins the gene and vector by forming phosphodiester bonds

-This produces a recombinant DNA molecule (plasmid + inserted gene)

how is recombinant DNA transferred into host cell [transforming cell]

the recombinant DNA is inserted into the host cell using electroporation

→it uses an electrical current that makes the membrane more permeable to allow the plasmid to pass through and enter cell

[Not all host cells take up the plasmid during transformation]

How are transformed cells identified?

using marker genes e.g. antibiotic-resistance markers, fluorescent markers, enzyme

How are transformed cells identified when the marker gene is on the plasmid?

→ antibiotic-resistance marker gene

→ fluorescent marker gene

antibiotic-resistance marker gene

-Plasmids contain an antibiotic-resistance marker gene (e.g. antibiotic resistance)
-After transformation, cells are grown on agar containing the antibiotic.
-Only cells that have taken up the plasmid survive because they are resistant→ these are the transformed cells

fluorescent markers

-Plasmids contain a fluorescent marker gene
-After transformation, cells are exposed to UV light.
-Only cells that have taken up the plasmid are fluorescent/glow

How are successfully transformed cells identified when the desired gene is inserted into a marker gene?

→plasmid has two genes for antibiotic resistance: to tetracycline and ampicillin→ desired gene is inserted into the gene for tetracycline resistance

-the desired gene disrupts the resistance to tetracycline

-cells are grown on agar containing ampicillin→ only cells that have taken up the plasmids survive and form colonies

-cells are grown on agar containing tetracycline→ cells with the desired gene cannot grow

-Therefore, cells that grow on ampicillin but not tetracycline contain the desired gene.

How are successfully transformed cells identified when the desired gene is inserted into a marker gene?

→desired gene is inserted into Green Fluorescent protein (GFP)

-the desired gene disrupts the GFP gene

-Cells are exposed to UV light

-the cells without the inserted gene are fluorescent (green) [are visible under UV light]

-the cells with the inserted gene are not fluorescent

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