Topic 21 - Recombinant DNA Technology Flashcards
What is recombinant DNA?
Introduction of a foreign gene into the DNA of another organism. The resulting organism is known as a genetically modified organism.
Why does DNA fragment from one organism, inserted into another, still produce the same protein?
The genetic code is universal, it’s the same in all organisms. Transcription and translation is also universal.
Method 1: Isolation of DNA fragments - Using reverse transcriptase to produce DNA from mRNA.
1) The mRNA has been transcribed from gene of interest.
2) Reverse transcriptase used to synthesise a single strand of complementary DNA (cDNA) from mRNA molecule.
3) DNA polymerase then forms the other strand of DNA from free nucleotides = double strand.
What is the advantage of using mRNA rather than DNA?
- Quantity: cells that make this protein will have lots of RNA.
- Searching: Don’t have to find gene on DNA in chromosome.
- The genetic code: Introns have already been removed.
Method 2: Isolation of DNA fragments using restriction endonucleases.
Restriction endonucleases found in bacteria that cut DNA at specific base sequences, so can be used to cut out a desired gene from the rest of genome.
Sticky ends:
- Cut ends of DNA, one strand longer.
- Have a strand of single stranded DNA, can attach to complementary DNA bases.
- Will join with another sticky end but only if cut with same RE.
Restriction Endonucleases:
- Highly specific active sites that catalyse hydrolysis of sugar-phosphate backbone of both strands of DNA molecule.
Why might some recognition sequences not be useful when isolating a particular gene?
If gene has recognition sequence in the middle, it’s useless as don’t want to cut the middle of the gene.
Method 3: Isolation of DNA fragments - fragments of DNA produced by creating gene in a ‘gene machine’ in a lab.
1) Desired nucleotide sequence derived from desired protein.
2) Nucleotide sequence fed into computer, which is checked. Small, single strands formed which assemble to form complete gene.
3) Oligonucleotides are created and joined to make gene.
4) Gene is replicated many times using PCR, makes gene into double-stranded DNA.
5) Using sticky ends, gene may be inserted into bacterial plasmid, acts as vector.
What is a plasmid?
Circular piece of DNA, separate from main bacterial DNA, contains only a few genes.
How is DNA prepared for insertion?
Before being inserted, extra lengths of DNA must be added to DNA fragment. These include:
- A promotor: length of DNA added before DNA fragment, to which transcriptional factors and RNA polymerase can bind to initiate transcription.
- A terminator: length of DNA added after DNA fragment, which causes RNA polymerase to be released and stop transcription.
Why are genes inserted into plasmids?
Plasmid acts as a ‘carrier’, or vector which can then be introduced back into a bacterial cell. Complimentary sticky ends makes this possible.
How is recombinant plasmids introduced into host cells?
1) Plasmids mixed with bacterial cells in ice-cold medium containing calcium ions.
2) Heat shock applied for 2 minutes. Bacterial cell membranes increase in permeability allowing plasmids to pass through into cell.
3) Only a few bacterial cells will take up plasmids, so gene markers can identify which bacterial cells have been successful in taking up plasmids.
What is the problem with introducing recombinant plasmids into host cells?
- Not all plasmids will take up the gene.
- Plasmids may join together.
- Recombinant DNA may join together.
Identification using gene markers: Antibiotic-resistance markers
Identification using gene markers: Fluorescent markers
1) Insert gene for a fluorescent protein into plasmid that also has the gene for ampicillin resistance.
2) Insert gene of interest into centre of a gene for a fluorescent protein.
3) Transfer plasmid into bacterial cell and grow in agar containing ampicillin.
4) Any bacterial cells that took up the plasmids will not be killed and will not be able to glow.
Identification using gene markers: Enzyme markers
1) Particular substrate that is usually colourless but turns blue when lactase acts upon it.
2) Insert chosen gene into gene that makes lactase, thereby inactivating the lactase gene. This plasmid contains gene for ampicillin resistance.
3) Transfer plasmid into bacterial cells.
4) Grow in agar containing ampicillin and the colourless substrate.
5) Any colonies that have taken up transformed plasmid will not be able to change its colour to blue.
6) Any colourless spots will indicate which cells have been transformed.
What is ‘in vivo’?
Performed in a living organism.
What is ‘in vitro’?
Performed in a test tube.
What is the polymerase chain reaction?
A method of copying or amplifying DNA fragments in the lab. It’s an automated process so is rapid and efficient.
What is needed for the polymerase chain reaction?
1) DNA fragments to be copied.
2) DNA polymerase to join together thousands of DNA nucleotides. Found in bacteria found in hot springs.
3) Primers to allow attachment of DNA polymerase and prevent two strands of DNA from joining.
4) Free DNA nucleotides.
5) Thermocycler to regulate and vary temperature over a period of time.
What are the stages of the polymerase chain reaction?
1) Strand separation - DNA heated at 95 degrees c for 5 minutes.
2) Mix with primers.
3) Annealing of primers - Mixture cooled to 55 degrees c.
4) Mix with free nucleotides and DNA polymerase.
5) DNA synthesis - Mixture heated to 70 degrees c (optimum for DNA polymerase)
6) Finish with 2 identical DNA molecules.
7) Repeats - With every cycle, the amount of DNA increases by 2x.
What are the advantages of using recombinant DNA technology as opposed to selective breeding to improve the productivity of crops and animals?
- Can move one gene at a time so more precise.
- Can make new genetic combinations not previously possible.
What are the advantages of recombinant DNA uses to humans?
- Increasing yield and nutritional value.
- Pest and disease resistant crops/animals.
- Crops resistant to herbicides.
- Crops resistant to extreme weather.
- Cultivating microorganisms for medicines.
- Production of active ingredients of medicines and vaccines.
What are some examples of genetically modified microorganisms?
- Bacteria and fungi naturally produce antibiotics and can be GMd to produce it in larger quantities.
- Enzymes required for food and beverage production. Brewing industry requires amylase to break down starch to produce glucose as energy source for yeast. Lipase in cheese production.
What are some examples of genetically modified plants?
- Tomatoes can be given complementary gene to block translation of softening gene when they ripen to transport.
- Rice crops developed to withstand infection of RBSDV virus and other viruses.
- Some plants given gene to produce toxins that are insecticidal - block respiratory pathways, paralysis.
