3.5 Flashcards
What is Gel electrophoresis?
a technique used to separate proteins or fragments of DNA according to its size. It can identify alleles at a few loci.
What does Gel electrophoresis use?
uses an electrical current to move molecules through a semisolid medium. The molecules are usually DNA, RNA or routines and they are separated according to their size and amount of charge.
How does DNA and RNA act in gel electrophoresis
DNA and RNA have a negative electrical charge and move towards the positive electrode in an electric field.
How are DNA molecules separated?
DNA molecules are often too long to be separated by electrophoresis. The appropriate size is usually 250-30 000 base pairs in length. To be cut down, DNA is digested by restriction endonuclease.
How do restriction endonuclease separate DNA?
These enzymes cut the backbone of DNA double helix at specific sequences to produce shorter DNA segments and distinctive fragment patters.
What are DNA patterns used to produce?
DNA profiles - combinations of DNA sequences are unique to each individual allowing anyone except identical twins to be identified by their DNA.
What takes place during electrophoresis?
Samples of fragments of DNA are loaded into wells on one end of the gel (a jelly-like polymer). The gel is submerged in a buffer solution and an electric current is run through the gel. The DNA samples must begin near the negative pole so that they spread out as they are drawn towards the positive pole.
What is the gel in gel electrophoresis like?
The consistency of the gel allows separation of DNA fragments by size. The gel is made of long polymers, often polysaccharide agarose, that binds together in an interwoven mesh or sieve. The DNA then has to travel between the spaces between the polymers. Smaller pieces can slip through the spaces more easily, allowing them to travel further along the gel in any given moment of time. Higher concentrations, means that the average size of the poor is reduced and smaller pieces of DNA can be separated.
What is a DNA ladder?
A DNA ladder is something which contains DNA fragments with a range of known lengths, by using it in the well, the length of the sample fragment can be determined.
What is polymerase chain reaction?
PCR is a technique that repeatedly copies fragments of DNA (between 100 and 40,000 base pairs) resulting in a large enough sample of DNA to do a thorough analysis
Where is DNA placed during PCR?
DNA is placed in a reaction chamber that contains many free nucleoside triphosphate, primers that will allow replication to occur from the desired point and a special heat stable version of DNA polymerase called Taq polymerase (originally found in bacteria that live in hot springs).
Why is Taq polymerase useful in PCR?
It does not denature at high temperatures used in PCR and can therefore continue to function in repeated cycles.
Describe the process of PCR
- DNA heated to break hydrogen bonds (98 degrees)
- The sample cools, short primer sequences will bond to complementary sequences in the DNA sample.
- The bonding of primers allows Taq polymerase to replicate DNA using the primer as a starting point.
- DNA is replicated
- DNA strands are heated to the point go separation and the process repeats.
If the starting DNA sample contains 10 ng of DNA, how much DNA will be present after five complete cycles of PCR?
320 ng ( each cycle doubles the DNA)
What are short repeated DNA sequences of the genome called?
Satellite DNA
What happens after amplifying DNA with PCR in DNA profiling?
Gel electrophoresis
What can DNA profiles determine?
Family relationships, paternity and murder suspects
name some examples of genetic modification
- Placing a bacterial insecticide gene into crops like potatoes
- Placing an antifreeze nee from an arctic fish into tomatoes
- Placing a spider-silly gene into goats
- Placing a gene for fluorescing glow proteins from a firefly into a tobacco plant
How is genetic modification carried out?
By the gene transfer of different species which is possible because of the universality of the genetic code.
Describe the process of gene transfer to bacteria
- Isolate the desired gene from the original species using restriction endonuclease. (reverse transcriptase needs to take place for eukaryotes)
- Isolate an appropriate plasmid
- Cut the plasmid with the same restriction endonuclease that was used to remove the desired gene to open the loop of the plasmid, forming a string on the two ends.
- Mix many copies of one target gene and cut plasmids together to allow their complementary unpaired sequences to join together, adding the gene int the plasmid.
- Use the enzyme DNA ligase to covalently bond the DNA backbones of the gene and plasmid together, permanently sealing the gene into the plasmid loop.
- Transfer the plasmid with the target DNA (recombinant plasmid) back into the bacterium
- Grow colonies of the genetically modified bacteria that now produce a eukaryotic protein.
Why are plasmids useful in genetic modification of bacteria?
They are easily transferable between bacterial cells and are small enough to manipulate easily.
What is used to seal a target gene into a bacterial plasmid?
DNA ligase
What questions would you ask to asses the risk/benefit of GMO (genetically modified organisms)
- How likely is a harmful or beneficial consequence
2. If the consequence occurs, how much harm or benefit will it cause?
Benefits of GMO crops
- Introduction of new positive traits to the crop: Golden corn (three added genes for more vitamins)
- Economic advantages - Bt corn (resistants to pests) produced 20-40% more corn per unit
- Environmental advantages: higher yields, less land is needed for forming Bt potatoes.
Risks of GMO
- Increases monoculture: corn ear worm and footwork pests have resistance to Bt in corn.
- Corporate control over food supply: Monsanto (GMO company) sues farmers for planting GM seeds from previous seasons. Increases inequality between traditional farms.
- Human health concerns: exposure to new genes may cause allergic reactions (anaphylaxis) was caused by GM starling corn.
What are clones?
Clones are groups of genetically identical organisms derived from a single original parent cell
Demonstrate a method of natural cloning in Bacteria
Binary fission in E.coli - the chromosome is copied and the cell splits in half, creating two cells each with a copy of the chromosome.
