- The mechanisms of Polymerase Chain Reaction
- DNA Profiling
- Genetic Transferal
Polymerase Chain Reaction (PCR) is a lab technique which takes a very small quantity of DNA and copies all the nucleic acids in it to make millions of copies of the DNA. The technique is used to get enough DNA to be able to analyze it.
1. Desired DNA is heated which breaks the hydrogen bonds between the strands of the double helix so that they separate.
2. Primers are added to start the process of DNA replication, and as the mixture is cooled, the primers bond to the original but now single stranded DNA molecules (through hydrogen bonding between complementary base pairs)
3. Nucleotides and a thermostable DNA polymerase is added. Nucleotides will bond with the "exposed" organic bases of the single stranded DNA. (again through H-bonding and CBP).
4. DNA polymerase will then join them into a DNA strand. Each original strand has formed a new complmentary strand. These strands are heated and seperated and function as a template for more DNA strands.
5. A large amount identical copies of the original DNA can be made exponentially.
Electrophoresis - technique used to separate large molecules based on their different rates of movement in an electric field caused by a combination of their charge and their size.
1. A gel is prepared into a thin layer and placed in a container. Holes are made on side where samples will be placed. (often at least one is an unknown sample)
2. Equipment is is connected to a source of electricity, and the gel will conduct electricity.
3. Depending on the charge of the molecules they will be more/less attracted to the other side. DNA fragments are slightly negative and will move towards the positive electrode. They will move through the gel, The speed they move depends on the attraction force and size of molecule. Large molecules move slower.
Gel electrophoresis is used in DNA profiling. It seperates section of DNA according to size and charge. This creates a pattern of stripes and bands determined by the sequence of organic bases. Every person's DNA is unique so it would be highly unlikely that 2 different people have the same pattern of DNA in the gel.
Technique for cloning
Reproductive cloning: creates a new individual (example: Dolly the sheep). First you take a nucleus from a somatic cell. The nucleus is removed from an egg cell and replaced with the nucleus from the somatic cell. A brief electric shock makes the cell start dividing. It grows into a group of cells, and is implanted into a uterus where it grows into a new individual with the same genetic material as the donated somatic cell.
A technique for cloning (Dolly):
1. From the donor, a non-gamete cell from the other was collected and cultured. The nucleus was removed.
2. An unfertilized egg was collected from another sheep and the nucleus was removed.
3. Using a zap of electrical current, the egg cell and the nucleus from the donor were fused together.
4. The new cell developed in-vitro in a similar way to a zygote, and started to form an embryo which developed normally.
Therapeutic cloning: involves stem cell research. Human embryos are produced and allowed to grow for a few days into a small ball of cells. These cells are not specialised and when SCNT is used the cells can grow into any of a large number different specialised tissues. Other sources of stem cells are cells from the umbilical cord or cells from aborted fetuses. This cloning is often aimed at cell therapy where diseased cells are replaced with healthy ones. Cell therapy is used for people suffering from Parkinson's disease but may also be possible when a patient has a spinal cord injury. Bone marrow transplants for patients with leukemia, new skin cells for burn victims and to grow new corneas for some visual impairments are examples of therapeutic cloning already in use.
Technique for gene transfer
Gene transfer involves the following elements:
- Vector to carry the gene into the host cell. (plasmids are very common)
- Host cell
- Restriction enzymes
- DNA ligase
Bacteria carry all required genetic info on one large circular DNA. They also possess extra DNA in the form of plasmids.
1. Splice the desired gene into a plasmid and transfer it into a bacterial cell.
2. Culture bacteria, many will have have a plasmid with the desire gene.
3. Use a section of nucelotides completementary to the desired genes but also attached to a radioactive label to find out which plasmids have the gene and which do not.
4. Use restriction enzymes to cut the desired gene out of the plasmids and purify the gene using gel electrophoresis.
Host cell - cell which recieves genetic information.
Restriction enzymes - used to cut a desired section of the DNA. Commonly used restriction enzymes recognizes the sequence GAATC and cuts both strands of the DNA between G and A.
- When prepping to transfer DNA, the same restriction enzyme is used for the host and the donor so the cuts are made in the say way.
- This means the same “sticky ends” are created so that the donor DNA can fit in between the host DNA.
And that's the end!
Drafted by Venetia (Biology)