Chapter 3.5 Flashcards
Uses of gel electrophoresis
It is used whenever the source of DNA needs to be identified. For example:
- to determine which strain of bacteria is causing an epidemic
- to solve crimes that hinge on DNA evidence.
- used to determine paternity and other family relationships.
What does gel electrophoresis do?
It is used to identify the alleles at a few (or a few dozen) loci.
What is needed to do gel electrophoresis?
It uses an electrical current to move molecules through a semisolid medium. The molecules, usually DNA, RNA or protein, are separated by their size and amount of charge.
Gel electrophoresis
a technique used to separate proteins or fragments of DNA according to size.
How is DNA cut for gel electrophoresis?
To get fragments of appropriate size, usually 250–30 000 base pairs (bp) in length, DNA is digested with special enzymes called restriction endonucleases. These enzymes cut the backbone of the DNA double helix at highly specific sequences, producing shorter DNA segments and distinctive fragment patterns
How is gel electrophoresis done? (1)
Samples with fragments of DNA are loaded into small depressions, called 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 can spread out as they are drawn toward the positive pole.
How is gel electrophoresis done? (2)
The consistency of the gel allows separation of the DNA fragments by size. The gel is made of long polymers, often the polysaccharide agarose, that bind together in an interwoven mesh or sieve. The DNA must travel through the spaces between the polymers. Smaller pieces can slip through the spaces more easily, allowing them to travel further along the gel in a given amount of time. By using higher concentrations of polymer, the average size of the pore can be reduced, and smaller pieces of DNA can be separated.
What does a DNA ladder do in gel electrophoresis?
Usually, one or more of the wells is filled with a ‘DNA ladder’, which contains DNA fragments with a range of known lengths. By using the DNA ladder, the length of sample fragments can be determined.
Why is PCR used?
In order to properly look at DNA identifiers, there needs to be enough DNA present to have a sample. However, when there is only a small sample, PCR is used to replicate it enough to make a proper sized sample for testing
What is used for PCR?
The desired section of DNA is placed in a reaction chamber that contains many free nucleoside triphosphates, 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). Taq polymerase is used because it does not denature at the high temperatures used in PCR and can therefore continue to function in repeated cycles.
Annealing
First, the DNA is heated enough to break the hydrogen bonds that hold the two strands of the double helix together. This occurs around 98 ºC. Then, as the sample is allowed to cool, the short primer sequences will bond (or anneal) to complementary sequences in the DNA sample
Replication
The bonding of primers allows Taq polymerase to replicate DNA using the primer as a starting point. (DNA polymerases are not able to add the first nucleotide of a DNA strand; they are only able to extend existing strands.) Once the DNA has been replicated, the DNA strands are heated to the point of separation and the process begins again.
Growth
Each time a cycle occurs, the amount of DNA doubles, resulting in exponential growth. Within a few hours, enough cycles of PCR have occured to create billions of copies of the DNA sequence. This provides ample copies for gel electrophoresis and other tests.
DNA profiling
a technique that examines variable portions of DNA to create a profile or ‘fingerprint’ that is unique to the individual.
Satellite DNA
short repeated DNA sequences
What is DNA profiling used for
- Forensics
- crime scenes
- determining familial relationships
- it can be used for species as well, such as biodiversity or strains of bacteria
Why can genes be transferred?
all living things share a common ancestor, the genetic code is essentially the same between species (universal). Therefore the information stored in DNA will be translated to the same polypeptide, whether it is read by a fungal, human, or any other cell.
How can genetic modification be done?
Some species have been modified by inserting the target gene into a virus and allowing it to infect the organism (viral vectors). Plants can be modified by shooting the cells with tiny gold pellets dipped into a solution containing the target gene (microprojectile bombardment). In all cases, the target gene is identified and isolated, then copied and inserted into the genome of a different species.
Steps for gene transfer for bacteria
- Isolate the desired gene from the original species using restriction endonucleases.
- Isolate an appropriate plasmid
- Cut the plasmid with the same restriction endonuclease that was used to remove the desired gene. This will open the loop of the plasmid, forming a string with two ends, most leave ‘sticky ends’ where one half of the helix extends beyond the other, leaving a few unpaired bases to be complementary
- Mix many copies of the target gene and cut plasmid together to allow their complementary unpaired sequences to join together.
- 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 (called a recombinant plasmid) back into the bacterium. - Grow colonies of the genetically modified bacteria that now produce a eukaryotic protein.
How are genes and plasmids in genetic modification complementary?
Using the same restriction endonuclease gives the gene and the plasmid complementary unpaired sequences that ‘stick’ together by hydrogen bonding.
How can you artificially make genes and plasmids complementary?
Use a restriction endonuclease that produces blunt ends for both gene and plasmid. Then add a series of guanine nucleotides to the 5’ ends of the gene, and cytosine nucleotides to the 5’ ends of the plasmid. This creates complementary sequences that will allow them to stick together.
Advantages of genetically modified crops
- introduction of new positive traits for the crops (increased vitamin content, drought or disease resistance, cold or salt tolerance, allergen reduction)
- economic advantages (longer shelf-life, less loss of food to environmental issues like disease, herbivores and frost; greater profit and lower prices for consumers.)
- enviromental advantages (Higher yields mean less land is needed for farming, leaving more natural ecosystem. Less pesticide needs to be sprayed.)
Disadvantages of genetically modified crops
- Ecosystem damage (outcompete native species, kill or damage non-pest species, or have other harmful effects. Cross-pollination could introduce the new genes to weed species.)
- Increasing monoculture (low biodiversity, little resistance if a new threat emerges.)
- Corporate control over food supply (Not all nations and farmers can access this technology. Increased pest attacks on traditional farms, and increased inequality between large farms and family or subsistence farms.)
- Human health concerns (Exposure to new genes and their protein products could cause unknown impacts and damage, allergic reactions and damage to mutualistic probiotic bacteria in the digestive system.)
Clones
Groups of genetically identical organisms, derived from a single original parent cell
Natural cloning advantages
The individuals do not need to find a mate, and they pass all their genetic information to each offspring. If an individual is well adapted and in a stable environment, its cloned offspring will have all the advantages of its parent.
Natural cloning in bacteria
Binary fission: The chromosome is copied and the cell splits in half, creating two cells each with a copy of the chromosome.
Natural cloning in Plants
Runners: Specialised stems grow along the ground and put down roots, creating a cloned individual a short distance from the parent plant.
Bulbs: Each clove in a head of garlic contains a cloned shoot of the parent plant as well as stored food to allow development of a new individual.
Tubers: Enlarged stems (potatoes) or roots (sweet potatoes) contain small buds called ‘eyes’, each of which can grow into a cloned plant.
Natural cloning in Fungus
Budding: The nucleus is copied and passed into a small bud formed on the side of the parent cell. The daughter cell is a clone, usually smaller than the parent cell.
Natural cloning in Animal
Budding: A new multicellular individual grows from the parent body using mitosis. When the cloned individual is large enough it breaks off.
Parthenogenesis: The adult lays eggs containing 100% of her genetic information. The eggs develop into clones. There are no males in this species.
Cloning in animals
- Splitting or fragmentation of an embryo to clone an animal before the cells have differentiated.
- Using differentiated cells and somatic cell nuclear transfer to clone adult animals.
Splitting or fragmentation of an embryo
For some animals, the early stage of an embryo is composed of cells that are totipotent stem cells. This means that each cell taken from that embryo has the potential to develop into any or all types of tissue. The cells can be individually separated and implanted into surrogate mothers. These ‘split’ embryos would then develop into clones of the original zygote and form normal organisms. This can happen spontaneously as well; monozygotic, or identical, twins in humans are born when a very early embryo breaks apart and each part develops independently.
Why can you not clone using differentiated cells?
In differentiated cells, some DNA has been inactivated during development, making it suitable for certain jobs but unable to perform others.
What are reasons to attempt to clone animals from differentiated somatic cells?
- to make copies of a genetically modified animal. GM animals require a large investment of time and resources to produce, and sexual reproduction would result in many offspring without the desired combination of traits.
- used to help prevent extinction by providing mates for critically endangered species.
Why can you not really clone humans or pets identically?
Just as identical twins are fully separate people with different interests and personalities, any clone would be a unique individual due to different environmental conditions and experiences.
Steps in somatic-cell nuclear transfer
- Donor somatic (body) cells are taken from the organism.
- Somatic cells with the least DNA inactivation are chosen.
- The cell is starved so that the amount of cellular material other than the nucleus is reduced.
- An unfertilised egg is taken from another individual.
- The unfertilised egg is enucleated (the nucleus is removed).
6.The enucleated egg is fused with a donor cell. - The fused cell is allowed to divide until a small embryo has formed.
- The embryo is transplanted into the uterus of a surrogate mother.
- The pregnancy and birth of the offspring proceed normally.
BT case and monarch butterflies
GM maize has been modified so that it produces a protein called Bt toxin from Bacillus thuringiensis bacteria. Bt toxin is lethal to the corn borer larvae of the moth, Ostrinia nubilalis, among other insects. Ideally, it allows farmers to apply less pesticide because the Bt toxin within the maize is already eliminating the corn borers. One concern about Bt maize was that the pollen of the maize also contains the Bt toxin. As maize is a wind-pollinated plant, its pollen is spread to surrounding areas including milkweed, the main source of food for monarch butterfly larvae. Initial reports, in 1999, suggested that in a laboratory setting monarch caterpillars were far more likely to die when feeding on milkweed plants that had been dusted with pollen from Bt maize than control milkweed plants. If the increased mortality was also true in the field, the GM maize crops posed a real threat to the survival of the monarch butterfly.However, in 2001, a report in the Proceedings of the National Academy of Science (PNAS) did not support the initial findings. In the actual milkweed fields, the concentration of pollen was rarely high enough to cause damage, and there were usually leaves with low concentrations of pollen that the larvae was likely to randomly select. Further, the monarch larvae were primarily damaged only when pollen release occurred early in their development. The research team found that the risk to monarchs from Bt toxin was minimal. Although the Bt crops may not be the primary cause, monarch butterfly numbers have dropped dramatically in the last 25 years. Possible reasons for the decline include climate change, habitat loss and destruction of milkweed by herbicides from neighbouring crop fields. Citizens along the migratory path of butterflies are attempting to protect the remaining populations by growing milkweed in their yards for larvae, collecting data for continuing studies and advocating for protected habitats.
Artificial cloning in plants
In certain plant species simply allowing a stem cutting to root by dipping the cut end in water will produce a clone. Some plants root easily when dipped in water or in a solid medium, while others need to be provided with an external source of rooting hormones.
internal plant factors affecting rooting
- Position of the cutting on the plant
- Age of the starting plant
- Nutritional status of the stem cutting
- Number or surface area of leaves on the stem cutting
external factors affecting rooting
- Length and intensity of light exposure
- Temperature at which the cutting is allowed to root
- Type and concentration of rooting hormones used
- Type and concentration of nutritional supplements used
- Type of growth medium (water, agar, soil, etc.)
