Genetic Engineering Techniques (OCR A Level Biology): Revision Note

Genetic Engineering Techniques

• In order for an organism to be genetically engineered the following steps must be taken:

  • Identification of the DNA fragment or gene

  • Isolation of the desired DNA fragment (either using restriction enzymes, a gene machine or reverse transcriptase)

  • Multiplication of the DNA fragment (using polymerase chain reaction - PCR)

  • Transfer into the organism using a vector (e.g. plasmids, viruses, liposomes). Electroporation is used to encourage uptake of plasmid vectors.

  • Identification of the cells with the new DNA fragment (by using a marker), which is then cloned

• Genetic engineers need the following 'tools' to modify an organism:

  • Enzymes

    • Restriction endonucleases - used to cut genes at specific base sequences (restriction sites). Different restriction enzymes cut at different restriction site

    • Ligase - used to join together the cut ends of DNA by forming phosphodiester bonds

    • Reverse transcriptase - Used to build double stranded DNA from single stranded RNA

  • Vectors - used to deliver DNA fragments into a cell

    • Plasmids - transfer DNA into bacteria or yeast

    • Viruses - transfer DNA into human cells or bacteria

    • Liposomes - fuse with cell membranes to transfer DNA into cells

  • Markers - genes that code for identifiable substances that can be tracked

    • Fluorescent markers e.g. green fluorescent protein (GFP) which fluoresces under UV light

    • Enzyme markers e.g. β-glucuronidase (GUS) enzyme which transforms colourless or non-fluorescent substrates into products that are coloured or fluorescent

    • Antibiotic resistance marker genes - The required gene sequence is inserted into a gene for antibiotic resistance. This inactivates the antibiotic resistance gene and therefore means that successfully transformed bacteria will be wiped out if exposed to the antibiotic. A replica plating method is then used to isolate the successfully transformed bacteria

An overview of the steps taken to genetically engineer an organism (in this case bacteria are being genetically engineered to produce human insulin)