Biotechnology

Question 1

What is gel electrophoresis, and what is its purpose?

Answer 1

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.

Question 2

Explain the reasoning behind why an electrical current is required for gel electrophoresis.

Answer 2

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.

Question 3

What is the DNA digested in gel electrophoresis digested with?

Answer 3

With restriction endonucleases, which cut the backbone of the DNA double helix at specific sequences, producing shorter segments and distinctive fragment patterns.

Question 4

Explain the process of DNA being separated in gel electrophoresis.

Answer 4

1. Samples with fragments of DNA are loaded into wells (small depressions) on one end of the gel.
2. The gel is submerged in a buffer solution, and an electric current is run through the gel.

Question 5

How does the consistency of the gel in gel electrophoresis allow for separation of DNA fragments by size?

Answer 5

–> 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

Question 6

What is a “DNA ladder”; how is it useful in gel electrophoresis?

Answer 6

A sample containing DNA fragments with a range of known lengths, allowing the length of other sample fragments to be determined.

Question 7

Why is loading dye necessary for gel electrophoresis? What dye is typically used?

Answer 7

The DNA fragments don’t have a colour. Ethidium bromide is commonly used because it binds to DNA and then fluoresces in UV light.

Question 8

What are some examples where gel electrophoresis can be used?

Answer 8

–> Detecting a strain of bacteria causing an epidemic
–> Solve crimes that hinge on DNA evidence
–> Determine paternity and other family relationships

Question 9

What does PCR stand for and what does it do?

Answer 9

Polymerase chain reaction; it repeatedly copies small DNA fragments, resulting in a large enough DNA sample to do a thorough analysis.

Question 10

How is PCR prepared? Why is Taq polymerase used?

Answer 10

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.

Question 11

Summarize the steps of PCR. (Does Anyone Else?)

Answer 11

1. Denaturation –> temperature increase (~98 C) to separate DNA strands by disrupting hydrogen bonds
2. Annealing –> temperature decrease (~60 C), allowing primers to base pair to complementary sequences on the DNA templates
3. 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

Question 12

What is DNA profiling?

Answer 12

A technique that examines variable portions of DNA to create a profile unique to the individual.

Question 13

Explain how DNA is obtained for DNA profiling.

Answer 13

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.

Question 14

What must be the condition for the DNA of an individual and another DNA sample to match?

Answer 14

The number and length of DNA fragments must be identical.

Question 15

How do you determine paternity through DNA profile?

Answer 15

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.

Question 16

State in simple terms how GMOs are created. Why is this possible?

Answer 16

By transferring DNA between species. All species have a common ancestor, so the genetic code is universal.

Question 17

Explain the seven steps of the process of gene modification. (Real Indie Rockers May Love Piano Concerts)

Answer 17

1. (Restriction the gene) – Isolate the desired gene from the original species using restriction endonucleases.
2. (Isolate plasmid) – Isolate an appropriate plasmid.
3. (Restriction the plasmid) – Cut plasmid with the same restriction endonuclease used to remove the desired gene.
4. (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.
5. (Ligase bonds the backbones!) – Use the enzyme DNA ligase to covalently bond DNA backbones of gene and plasmid together.
6. (Plasmid back to bac) – Transfer the recombinant plasmid back into the bacteria.
7. (Colonies create a eukaryotic protein!) – Grow colonies of the genetically modified bacteria that now produce a eukaryotic protein.

Question 18

How does the process of isolating a gene from its original species change when the gene is eukaryotic, and why?

Answer 18

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.

Question 19

How do restriction endonucleases usually facilitate the joining of the gene and plasmid?

Answer 19

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.

Question 20

Explain two major benefits of GMO crops, and give an example for each.

Answer 20

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.

Question 21

Explain two major risks of GMO crops, and give an example for each.

Answer 21

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

Question 22

Define clones.

Answer 22

A group of genetically identical organisms, derived from a single original parent cell.

Question 23

What mode of reproduction is likely to produce clones? Explain why.

Answer 23

Asexual reproduction.

Bacteria divide by binary fission, so each resulting bacterium is an identical copy of the other (excepting any mutations).

Question 24

What are the advantages of natural cloning?

Answer 24

• 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

Question 25

Explain one method of natural cloning for plants, and one for animals.

Answer 25

Four answers for plants, two for animals below

Runners (plants) – specialized stems grow along ground and put down roots, cleating a clone a short distance from the parent plant

Bulbs (plants) - each lobe of a plant bulb contains a cloned shoot of the parent plant and stored food

Tubers (plants) - enlarged stems/roots contain small buds that can grow into a cloned plant

Budding (plants) - nucleus is copied and passed into a small bud formed on the side of the parent cell

Budding (animals) - a new multicellular individual grows from parent body using mitosis –> once large enough it breaks off

Parthenogenesis (animals) - adult lays eggs containing 100% of genetic info, that develop into clones

Question 26

What are the two different methods of deliberately cloning animals that scientists have developed?

Answer 26

1. Splitting/fragmenting an embryo to clone an animal before the cells have differentiated.
2. Use differentiated cells / somatic cell nuclear transfer to clone adult animals.

Question 27

Explain the process of splitting/fragmenting an embryo.

Answer 27

–> In some animals, early stage of an embryo is composed of totipotent stem cells.
–> You can split an eight-cell embryo into eight individual cell and implant each into a surrogate mother
–> The “split” embryos will develop into eight clones of the original zygote and form normal organisms

(Note: Identical twins are a natural example of this–an early embryo breaks apart and each part develops independently)

Question 28

Explain the six steps of somatic-cell nuclear transfer

Answer 28

1. (Extract SBC) Donor somatic (body) cells are taken from the organism that will be cloned and cultured in the lab.
2. (Extract egg) An unfertilized egg is taken from another individual.
3. (Enucleation) The unfertilized egg has its nucleus removed.
4. (Fuse!) The enucleated egg is fused with a donor cell.
5. (Division) The fused cell is allowed to divide until a small embryo has formed.
6. (Transplant) The embryo is transplanted into the uterus of a surrogate mother.

Question 29

What was the concern of Bt maize with regards to its impacts on monarch butterflies?

Answer 29

The pollen of the maize contained the Bt toxin, which was spread to surrounding areas including milkweed, the main source of food for monarch butterfly.

Lab reports in 1999 suggested that monarch caterpillars were more likely to die when feeding on milkweed plants dusted with pollen from Bt maize.

Question 30

What were the conclusions made with the study of the negative impact of Bt maize upon monarch butterflies?

Answer 30

A report in 2001 did not support initial threatening findings.
–> In actual milkweed fields, the concentration of pollen was usually too low to cause damage
–> Larvae often selected leaves with low concentrations of pollen
–> Larvae were primarily only damaged when pollen release occurred early in their development
Ultimately, it was concluded that the risk to monarchs due to Bt toxins was minimal.

Question 31

What is the name of the bacterium that produces the Bt toxin? (who cares >:| ?????)

Answer 31

Bacillus thurigiensis.

Question 32

What is rooting of stem-cuttings?

Answer 32

It’s an artificial cloning method that is possible with certain plant species. You cut the step and then allow it to root by dipping the cut end in water, and a clone is produced.

Question 33

Name two internal plant factors and two external plant factors that affect rooting.

Answer 33

Four internal examples and five external examples below!

Internal:
–> Position of cutting on the plant
–> Age of the starting plant
–> Nutritional status of the stem cutting
–> Number or surface area of leaves on stem cutting

External:
–> 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.)

Question 34

Outline how you would design an experiment on rooting of stem-cuttings.

Answer 34

1. Select an independent variable to investigate (factors affecting rooting), as well as a range and five levels to test.
2. Select a quantitative measurement as the dependent variable (success of rooting)
3. Write a research question: How does independent variable affect rooting of stem cuttings as measured by dependent variable
4. Provide a brief method focusing on controlled variables (any of the external or internal variables other than the IV) and repeat trials
5. Describe how data will be collected and processed (extracting data to graph, performing a statistical test)

Question 35

What is non-coding DNA defined as?

Answer 35

DNA sequences within a genome that do not consist of the information to make a protein and are never represented within the amino acid sequence of expressed protein.

Question 36

Explain the four regions of DNA that do not code for proteins.

Answer 36

Regulators of gene expression:
–> Promoters – sequences occurring just before genes that act as a binding point for RNA polymerase enzymes
–> Enhancers/silencers – binding sites for proteins that either increase or decrease rate of transcription

Introns:
–> DNA base sequences found within eukaryotic genes that are removed at the end of transcription

Telomeres:
–> Repetitive sequences that protect the ends of the chromosome, ensuring that DNA is correctly replicated
–> Short stretches lost from telomeres every cell division

Genes for tRNAs:
–> Code for RNA molecules that don’t get translated into proteins, but fold to form tRNA molecules for translation

Question 37

What is a tandem repeat? Where are they usually found?

Answer 37

A sequence of two or more DNA base pairs that is repeated in such a way that the repeats lie end-to-end on the chromosome.

They generally form part of non-coding DNA.

Question 38

What type of tandem repeats are used for identification in DNA fingerprinting? Why are they used?

Answer 38

Those that are located at a single genetic locus, in which the number of repeated DNA segments varies from individual to individual.

Because of that variance, differences in these regions can be analyzed to produce a DNA profile.