Polymerase chain reaction
DNA heated to 95 degrees to denature DNA - strands seperate as hydrogen bonds are broken.
Cooled to 55 degrees and primer DNA added
At 70 degrees, Taq polymerase binds to the primer. This synthesises identical strands of DNA by adding on free nucleotides
Cycle is repeated many times. Each cycle doubles the amount of DNA present.
- DNA chopped into fragments by Restriction endonuclease enzymes
- PCR used to multiply amount of DNA present
- Agarose gel created, and an electric current is set up across it. DNA is put into wells at the negatively charged end, along with a negatively charged dye
- The current is stopped when the dye reaches the positive electrode. Smaller fragments will have travelled further than larger fragments
- Southern blotting used to transfer the DNA to a nylon membrane:
- 1. Washed in a basic solution to seperate 2 strands of DNA
- 2. Washed in a salt solution
- 3. Nylon membrane pressed onto gel - DNA transfers onto membrane.
- A radioactive probe is added, which binds to the DNA
- X-rayed = DNA shows up as dark lines
- Desired band identified and cut out of gel. This DNA is multiplied using PCR
Sequencing = working out order of bases in one chain of DNA
- DNA multiplied using PCR and broken into fragments using restriction enzymes
- Primers attach to the 3' end. DNA Polymerase binds to the primer
- DNA Polymerase adds complementary nucleotides until a chain terminator joins
- Fragment is released
- Repeated many times to make all possible fragment lengths
- Gel electrophoresis used to seperate fragments
- Fragments passed through a tube into a computer, which can work out the sequence of bases in the DNA.
Genetic engineering of bacteria 1
Step 1: Gene extracted using Restriction enzymes:
- The enzymes (from microorganisms) cut DNA at specific nucleotide sequences, which are called recognition sites. Enzymes are selected that will cut either side of the desired gene
- Gel electrophoresis is used to identify which gene is needed
- PCR is used to make copies of the gene
Stage 2: Inserting the gene into a vector:
- The vector is normally a plasmid removed from a bacterial cell
- Plasmid cut open using the same restriction enzymes to produce complementary sticky ends
- The gene is added with some DNA ligase to make the sticky ends stick together.
Genetic engineering of bacteria 2
Stage 3: Getting the plasmids into bacteria:
- Cells treated with Calcium ions to increase permeability of cell membranes
How do you know if bacteria has taken up plasmid, and if the plasmid has the gene inserted?
1. Replica plating - 2 genes for antibiotic resistance.
- Bacteria put on agar containing ampicilline, Only those with a plasmid will have resistance and will grow
- Surviving bacteria put on agar containing tetracycline. Gene for tetracycline resistance cut open, so those with the desired gene will not grow as they will no longer have resistance to tetracycline.
2. Fluorescence. Successfully genetically modified bacteria grown in a fermenter and proteins produced collected and purified.
Golden Rice - genetically modified plants
Designed to prevent Vitamin A deficiency, which can cause blindness
- 1. Gene for beta carotene production extracted from daffodils using bacterium Erwinia uredovora
- 2. Gene extracted and inserted into a plasmid
- 3. Plasmids inserted into a bacteria called Agrobacterium tumefaciens
- 4. These bacteria naturally infect plants, so could introduce the GM plasmid into the rice cells
- 5. Bacteria mixed with rice embryos in petri dishes. Some embryos infected by bacteria carrying gene for Beta carotene production
- Rice embryos grown into adult plants. Produced seeds containing beta carotene.
Advantages and Disadvantages of Golden Rice
- Helped combat blindness
- Everyone needed to grow golden rice
- Unknown long-term effects
- Pollen is transported long distances, so cannot be controlled
- May not have a high enough yield or taste as good as normal rice.
Cross breeding was necessary to get a high Beta-carotene content as well as a high-yielding variety of crop.
- Pig organs are of a similar shape and size to human organs
- Pig organs would trigger an immediate immune response, and would therefore be rejected
- Most of the immune response is targetted at a certain glycoprotein in the pig cell membrane
- Pigs can be genetically engineered not to contain the GGTA1 gene
Problems with xenotransplantation:
- Is it morally right?
- Pig diseases could evolve and be able to pass to humans
- Immune response may still be large enough to cause rejection
- Pigs don't live as long as us, so will their organs last as long?
- The use of pigs is against some religions
Involves altering alleles to cure genetic disorders.
If caused by 2 recessive alleles - introduce a dominant allele to overrule the two recessive alleles.
If caused by a dominant allele - Silence the allele by putting a bit of DNA through the middle of the allele's gene.
Somatic therapy - modification of normal body cells. eg. in CF, epithelial cells lining the lungs were targetted. Offspring may still inherit the disorder
Germ-line therapy - modification of gametes. Offspring will not have the disorder. Currently illegal.