1) Denaturation - DNA sample is heated to separate into two strands.
2) Annealing - DNA primers attach to opposite ends of target sequence.
3) Elongation - Heat tolerant DNA polymerase (Taq) copies the strands.
Gel electrophoresis of DNA
In gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size.
1) Sample of fragmented DNA is placed in walls of an agarose gel.
2) Gel placed in buffering solution/electrical current passed across gel.
3) DNA's charge goes to positive.
4) Fragments are then separated according to size.
Gel electrophoresis in DNA profiling
Gel electrophoresis of DNA is used in DNA profiling.
1) Satellite DNA (repeating sequences are short tandem repeats).
2) These repeats are excised to form fragments by cutting a variety of restriction endonulceases (those that cut DNA at specific sites).
3) Individuals have different number of repeats, and are unique.
Application of DNA profiling to determine paternity and in forensic investigations
1) DNA sample collected and amplified by PCR.
2) Satellite DNA (non-coding) is cut with specific restriction enzymes to generate fragments.
3) Individuals have unique fragment lengths due to variable length of short tandem repeats (STR).
4) Fragments separated with gel electrophoresis.
5) DNA profile can be analyzed; 2 applications - paternity testing and forensic investigations.
Analysis of DNA profiles
Paternity analysis (science.howstuffworks.com)
Three outcomes of human genome project
Mapping has allowed us to know number, location and basic sequence of human genes.
Screening has led to production of specific gene probes to detect sufferers and carriers of genetic disease conditions.
Medicine has seen discovery of new proteins and their functions, from which we can develop improved treatments (pharmacogenetics/rational drug design).
Ancestrally, we have improved insight into our origins, evolution and historical migratory patterns of humans.
We can also begin sequencing of non-human organisms.
Universal genetic code
When genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal.
Genetic code is universal, every living organism has the same codons for same amino acids; therefore, genetic info could be translated by another.
Basic technique used for gene transfer
DNA extraction
1) Plasmid removed from bacterial cell (plasmids are small, circular DNA molecules that can exist and replicate autonomously)
2) Gene of interest removed from an organism's genome using a restriction endonuclease which cut at specific sequences of DNA.
3) Gene of interest and plasmid are both amplified by PCR.
Digestion and Ligation
1) Plasmid cut with same restriction enzyme that was used to excise the gene of interest.
2) Generates overhangs (stick ends) allowing two DNA constructs to fit together.
3) Gene of interest and plasmid spliced together by DNA ligase, creating a recombinant plasmid.
Transfection and Expression
1) Recombinant plasmid inserted into desired host cell.
2) Transgenic cells hopefully produce desired trait encoded by gene of interest (expression).
3) Product may need be isolated from host and purified in large amounts.
Two examples of the current uses of genetically modified crops or animals
Salt tolerance in tomato plants.
Synthesis of beta-carotene (vitamin A precursor) in rice.
Herbicide resistance in crop plants.
Factor IX (human blood clotting) in sheep milk.
Potential benefits and possible harmful effects of one example of genetic modification
| Herbicide Tolerant Crops | |
| Benefits | Dangers |
| Eradicates weeds, loss of yield due to competition with weed. | Fewer weed species on farmland, and longer periods without weeds growing, breaks wildlife food chain. |
| Glysophate herbicide transported all around weed plant kills all weeds even those with extensive roots. | Selection of glysophate resistant rops ties the grower to one particular herbicide product - choice is lost. |
| Glysophate inhibits nzyme for production of amino acids in plants; enzyme absent from animals, lowering toxicity. | Will GM plant material, when consumed by humans, release novel toxins or otherwise adversely affect enzyme systems in human digestive system? |
| Herbicide applied as large droplets from coarse nozzles, rather than as a fine mist of tiny droplets that are prone to drift onto surrounding habitats. | If glyphosate herbicide droplets do reach hedgerow plants, their size/chemical activity in plant means they are more likely to do damage. |
Group of genetically identical organisms or a group of cells derived from a single parent cell.
Technique for cloning using differentiated animal cells
Somatic cell nuclear transfer
1) Female animal treated with hormones (FSH) to stimultae egg development.
2) Nucleus from egg removed (enucleated), removing genetic info from cell.
3) Egg fused with nucleus from a somatic (body) cell of another sheep, making egg cell diploid.
4) Electric shock delivered to stimulate egg to divide, once process begins, egg implanted into uterus of surrogate.
5) Developing embryo has same genetic material as that sheep that contributed the diploid nucleus, thus a clone.
Ethical issues of therapeutic cloning in humans
| Therapeutic Cloning | |
| For | Against |
| Used to cure serious diseases with cell therapy. | Involves creation and destruction of human embryos. |
| Stem cell research paves way for future discoveres (replace bad cells with good ones) and beneficial technologies tht would not occurred if use was banned. | Embryonic stem cells are capable of continued division and may cause and develop into cancerous cells and cause tumors. |
| Parents at high risk of producing offspring with genetic disease could have healthy children (infertile couples too). | Human beings might be planned and produced with sole intention of supplying spare parts for a related human being (with health problems). |
| Cloning techniques are safe and reliable as other comparable medical procedures. | Techniques are experimental and unreliable because there are many deaths of embryos and new borns. |
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