Biotechnology Flashcards
(38 cards)
What is gel electrophoresis, and what is its purpose?
It is a technique that can identify alleles at a few or few dozen loci. It uses an electrical current to separate biomolecules (e.g. DNA, RNA) in agarose gel (a semisolid medium) according to their size and electrical charge.
Explain the reasoning behind why an electrical current is required for gel electrophoresis.
DNA and RNA molecules have a negative electrical charge (phosphate group PO43-) and will move from the cathode to the anode in an electric field, facilitating movement that allows for identification.
What is the DNA digested in gel electrophoresis digested with?
With restriction endonucleases, which cut the backbone of the DNA double helix at specific sequences, producing shorter segments and distinctive fragment patterns.
Explain the process of DNA being separated in gel electrophoresis.
- Samples with fragments of DNA are loaded into wells (small depressions) on one end of the gel.
- The gel is submerged in a buffer solution, and an electric current is run through the gel.
How does the consistency of the gel in gel electrophoresis allow for separation of DNA fragments by size?
–> The gel is made of long polymers (usually agarose) that bind together into a mesh
–> The DNA has to travel through the spaces between the polymers
–> Small pieces slip through the spaces more easily and can travel faster along the gel
–> High concentrations of polymer can reduce the average size of the pore, separating smaller pieces of DNA
What is a “DNA ladder”; how is it useful in gel electrophoresis?
A sample containing DNA fragments with a range of known lengths, allowing the length of other sample fragments to be determined.
Why is loading dye necessary for gel electrophoresis? What dye is typically used?
The DNA fragments don’t have a colour. Ethidium bromide is commonly used because it binds to DNA and then fluoresces in UV light.
What are some examples where gel electrophoresis can be used?
–> Detecting a strain of bacteria causing an epidemic
–> Solve crimes that hinge on DNA evidence
–> Determine paternity and other family relationships
What does PCR stand for and what does it do?
Polymerase chain reaction; it repeatedly copies small DNA fragments, resulting in a large enough DNA sample to do a thorough analysis.
How is PCR prepared? Why is Taq polymerase used?
The desired DNA section is placed in a reaction chamber containing:
–> free nucleoside triphosphates
–> primers that allow replication to occur from the desired point
–> Taq polymerase
Taq polymerase is used because it does not denature at PCR temperatures and continues to function in repeated cycles.
Summarize the steps of PCR. (Does Anyone Else?)
- Denaturation –> temperature increase (~98 C) to separate DNA strands by disrupting hydrogen bonds
- Annealing –> temperature decrease (~60 C), allowing primers to base pair to complementary sequences on the DNA templates
- Extension –> Heat resistant Taq polymerase binds to the primer sequences and adds nucleotides to extend the second strand –> process is repeated, region of interest is increased exponentially
What is DNA profiling?
A technique that examines variable portions of DNA to create a profile unique to the individual.
Explain how DNA is obtained for DNA profiling.
Restriction endonucleases are used to chop satellite DNA (short repeated DNA sequences) into fragments of varying length. The fragments are amplified with PCR, and the resulting mix of DNA fragments is separated using gel electrophoresis.
What must be the condition for the DNA of an individual and another DNA sample to match?
The number and length of DNA fragments must be identical.
How do you determine paternity through DNA profile?
Match DNA fragments from the child with the mother’s profile. Every band of the child’s DNA that does not match the mother must have a match in the father’s profile.
State in simple terms how GMOs are created. Why is this possible?
By transferring DNA between species. All species have a common ancestor, so the genetic code is universal.
Explain the seven steps of the process of gene modification. (Real Indie Rockers May Love Piano Concerts)
- (Restriction the gene) – Isolate the desired gene from the original species using restriction endonucleases.
- (Isolate plasmid) – Isolate an appropriate plasmid.
- (Restriction the plasmid) – Cut plasmid with the same restriction endonuclease used to remove the desired gene.
- (Mixing and joining the comp unpaired) – Mix many copies of the target gene and cut plasmid together to allow their complementary unpaired sequences to join together.
- (Ligase bonds the backbones!) – Use the enzyme DNA ligase to covalently bond DNA backbones of gene and plasmid together.
- (Plasmid back to bac) – Transfer the recombinant plasmid back into the bacteria.
- (Colonies create a eukaryotic protein!) – Grow colonies of the genetically modified bacteria that now produce a eukaryotic protein.
How does the process of isolating a gene from its original species change when the gene is eukaryotic, and why?
You use reverse transcriptase to produce an edited version of the gene.
Eukaryotes delete parts of the RNA before it leaves the nucleus to be translated, but bacteria don’t have a nucleus and can’t perform this step. Scientists compensate by making DNA without the parts that should be deleted.
How do restriction endonucleases usually facilitate the joining of the gene and plasmid?
Most restriction endonucleases leave “sticky ends” – one half of the helix extends beyond the other, leaving a few unpaired bases. Using the same restriction endonuclease for the gene and plasmid give them complementary unpaired sequences.
Explain two major benefits of GMO crops, and give an example for each.
Three possible answers below!
Introduction of new positive traits to a crop – increased vitamin content, drought/disease resistance
Example: golden corn –> adds genes to provide vitamin A precursors, preventing blindness caused by vitamin A deficiency
Economic advantages – longer shelf-life, less loss of food to disease/herbivores/frost
Example: Bt corn (added gene to resist pests) – can produce 20%-40% more corn per unit of land
Environmental advantages – less land needed for farming, less pesticide needed
Example: documented drop in sprayed pesticide on farms using Bt potatoes.
Explain two major risks of GMO crops, and give an example for each.
Four possible answers below!
Ecosystem damage – outcompeting native species, killing/damaging non-pest species, harmful cross pollination
Example: GM creeping bentgrass hybridized with wild grasses, spreading the new gene into the wild
Increasing monoculture – GMO crops have low biodiversity (cloned from an original modified plant). Little resistance if a new threat emerges
Example: Corn earworm / rootworm pests have developed resistance to Bt in corn
Corporate control over supply – increased pest attacks on traditional farms, increased inequality between large farms and family/subsistence farms
Example: Some subsistence farmers near large GMO farms have high numbers of pests in their fields
Human health concerns: possible allergic reactions, possible damage to mutualistic probiotic bacteria
Example: trying to improve nutrition of soybeans with a Brazil nut protein made people allergic to the nuts allergic to the soybeans
Define clones.
A group of genetically identical organisms, derived from a single original parent cell.
What mode of reproduction is likely to produce clones? Explain why.
Asexual reproduction.
Bacteria divide by binary fission, so each resulting bacterium is an identical copy of the other (excepting any mutations).
What are the advantages of natural cloning?
- Individuals do not need to find a mate
- They pass all their genetic information to each offspring
- The clone of a well-adapted offspring in a stable environment will have all the advantages of its parent
