6.3.9 Genetic Engineering Techniques Flashcards
What are the steps needed for an organism to be genetically engineered?
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
What tools can modify organisms?
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
