Chapter 2: DNA Manipulation Techniques

Question 1

Define enzyme

Answer 1

• Enzymes are biological catalysts that speed up reaction rates by reducing activation energy

Question 2

Define endonuclease

Answer 2

• An endonuclease, also known as a restriction enzyme, is an enzyme that cuts DNA at specific restriction/recognition sites
• Also known as “molecular scissors”

Question 3

Compare sticky and blunt ends

Answer 3

• Sticky ends are ends of a DNA fragment with overhanging base pairs after being cut by an endonuclease
  
  • Allow foreign DNA to be inserted into the genome with greater control
  • Pair faster compared to blunt ends
• Blunt ends are ends of a DNA fragment with no overhanging base pairs after being cut by an endonuclease
  
  • Scientists have less control over the insertion of foreign DNA

Question 4

Define DNA polymerase

Answer 4

• DNA polymerase is an enzyme involved in DNA synthesis

Question 5

Define DNA ligase

Answer 5

• DNA ligase is an enzyme that connects DNA together by catalysing the formation of phosphate bonds

Question 6

Describe CRISPR

Answer 6

• Clustered regularly interspaced short palindromic repeats
• An immune response found naturally in bacteria
• Provides bacteria with natural immunity against viral attacks

Question 7

Define bacteriophage

Answer 7

• Bacteriophages are viruses that infect and replicate only in bacterial cells

Question 8

Define the role of Cas9

Answer 8

• Cas9 is an endonuclease that seeks and destroyes DNA indentical to the copied segments of DNA (spacers) from previous invasions

Question 9

Outline the process that CRISPR-Cas9 uses to cut specific DNA

Answer 9

• A bacteriophage attaches outside a bacterial cell and injects its viral DNA into the cell (via previous invasions, a segment of the viral DNA has been stored as a spacer in the CRISPR region)
• The CRISPR sequence is transcribed resulting in CRISPR RNA (crRNA)
• Tracer RNA (trcrRNA) has a complementary sequene to repeat DNA
• crRNA and tracer RNA (tcrcRNA) form guide RNA (gRNA)
• gRNA binds with cas9 forming a cas9-gRNA complex
• Cas9-gRNA complex scans bacteriophage (target DNA) and looks for complementary bases and a PAM sequence
• The target DNA (bacteriophage) is unzipped
• Cas9 cuts/cleaves the DNA several nucleotides upstream of the PAM sequence
• The viral DNA has been disrupted and therefore cannot reproduce

Question 10

Describe how CRISPR-Cas9 can be programmed to edit a genome

Answer 10

• Scientists create a sgRNA-Cas9 (or crRNA-Cas9) complex with an RNA sequence that is complementary to the target DNA sequence
• Cas9 recognises the PAM sequence and identifies complementary bases via the specific sgRNA
• Cas9 cuts/cleaves the DNA several nucleotides upstream of the PAM sequence
• Scientists insert, remove or replace sections of DNA
• The cell recognises and repairs the broken DNA causing any changes to become integrated into the genome

NOTE: Naturally, guide RNA is complementary to the spacer (DNA from virus). In the lab, we can create sgRNA that is complementary to a gene of interest (allows CRISPR to target any DNA sequence).

Question 11

Describe the difference between Cas9 and typical endonucleases

Answer 11

• Unlike typical restriction enzymes, Cas9 cannot just cut any complementary DNA sequence, it requires a PAM sequence adjacent to the target site in order to cleave
• Typical restriction enzymes cut at a set recognition site sequence, whereas Cas9 cuts at a sequence that is designated by guide RNA

Question 12

Outline the ethical concerns associated with CRISPR-Cas9

Answer 12

• Respect → embryos cannot give informed consent
• Respect → some believe that modifying embryos does not honour the sanctity of life
• Justice → gene editing technology is expensive and may only be available to wealthy people
• Non-maleficence → treated individuals or embryos may experience unforeseen side effects

Question 13

Describe the difference between gRNA and sgRNA

Answer 13

• Naturally, gRNA (guide RNA) is comprised of 2 RNA molecules: crRNA and tracrRNA
• In the lab, scientists manufacture a synthetic sgRNA (single guide RNA) that is comprised of 1 RNA molecule
  
  • sgRNA serves as both the tracrRNA and crRNA (containing the spacer)

Question 14

Explain why using CRISPR to correct inherited genetic diseases is mainly suggested for embryos and not adults

Answer 14

• Any change that is made to an embryo is integrated into its genome as the embryo grows and as its cells replicate and differentiate
• Changes made to an adult’s genes will only affect the cells that are directly edited

Question 15

Define and describe the importance of PAM sequences

Answer 15

• Protospacer adjacent motif (any nucleotide followed by GG)
• Allows bacteria to determine self from non-self
• Cas9 cuts only where there are complementary bases AND a PAM sequence
• This ensures that Cas9 doesn’t cut the bacteria’s own genome
  
  • Spacers are identical to the target viral DNA but do not have a PAM sequence, therefore, they are recognised as self and are not cut by Cas9

Question 16

Identify 2 ways in which the CRISPR-Cas9 system is similar to the human immune system

Answer 16

• CRISPR-Cas9 provides bacteria with an immunological memory of specific pathogens just as the human immune system has immunological memory due to having B memory cells
• The CRISPR-Cas9 system responds to antigens by secreting a protein (Cas9) that cuts the foreign DNA and the human immune system also responds to antigen by secreting proteins

Question 17

State the purpose of PCR

Answer 17

• Used to amplify DNA
• Amount of DNA doubles after each cycle

Question 18

Outline the process of polymerase chain reaction (PCR)

Answer 18

• Denaturing → DNA is separated into two strands by heating it at 94°C
  
  • The heat breaks the hydrogen bonds between the complementary DNA strands
• Annealing → DNA is cooled at 54°C which allows primers to bind to the DNA
  
  • Primers are segments of single-stranded DNA that initiate DNA synthesis
  • Two primers are needed as they define (or “frame”) the section of DNA that needs to be amplified (one binds at the start and the other binds at the end of the target DNA)
• Extending → DNA is heated at 72°C to allow Taq polymerase (DNA polymerase enzyme) to extend the primers using free nucleotides to form two complete double strands
  
  • A supply of free nucleotides must be available for Taq polymerase to create a new strand of complementary DNA

NOTE: Denaturing must occur first to ensure that DNA is single stranded. This allows primers to bind to each strand.

Question 19

Explain why Taq polymerase is suitable to be used in PCR

Answer 19

• Taq polymerase has an optimal temperature of 72°C
• When the DNA sample is heated at 94°C, Taq polymerase does not denature
• This allows Taq to catalyse the formation of a new DNA strand

Question 20

Define short tandem repeats (STRs)

Answer 20

• Chromosomal sites in nuclear DNA where multiple copies of a short DNA sequence are repeated and joined end to end

Question 21

Outline the use of STRs

Answer 21

• Can be used for DNA profiling
  
  • Establishing the identity of missing people
  • Confirming familial relationships
  • Linking suspects to crime scene evidence
• The number of repeats in STR markers vary significantly among individuals making them effective for human identification purposes

Question 22

Explain how gel electrophoresis can separate DNA based on size

Answer 22

• DNA is negatively charged
• When placed in an electric field, DNA fragments will move toward the positively charged pole of the gel
• Smaller DNA strands move through the gel faster than larger strands
• Hence, fragments will be arranged in order of size

NOTE: DNA fragments further from the origin are smaller. DNA fragments closer to the origin are larger.

Question 23

Outline the process of ‘running a gel’

Answer 23

• DNA sample with fragments of varying sizes placed in wells on the negatively charged end of the machine
• Electricity applied to the gel
• DNA is attracted to the positively charged end
• Smaller fragments move through the gel faster than larger fragments
• Fragments appear as bands on the gel which can then be interpreted (by comparing with the standard)

NOTE: Standards are DNA fragments of known sizes that allow for the approximation of unknown sized fragments

Question 24

Explain why blunt ends are preferred over sticky ends when performing gel electrophoresis

Answer 24

• Hydrogen bonds between base pairs may reform for DNA with sticky ends
• Sticky ends cause fewer bands on the gel producing less reliable results

Question 25

Compare genetically modified and transgenic organisms

Answer 25

• Genetically modified organisms (GMO) are organisms that have had their DNA directly manipulated through genetic manipulation technology
  
  • E.g. Flavr savr tomato (specific genes silenced)
• Transgenic organisms are organisms that are also modified and have DNA from a different species
  
  • E.g. GloFish (include DNA from a sea anemone)

Question 26

Define plasmid

Answer 26

• Plasmids are circular DNA found in prokaryotes

Question 27

Define recombinant plasmids

Answer 27

• Plasmids that carry foreign DNA

Question 28

Outline the process of producing recombinant plasmids

Answer 28

• Plasmid is cut using a specific endonuclease to create sticky ends
  
  • Plasmid goes from circular to linear
• Foreign DNA is cut using the same endonuclease
  
  • Sticky ends are complementary to the plasmid because the same endonuclease was used
• Foreign DNA and plasmid is mixed
  
  • Initially (in some cases), their sticky ends join weakly via hydrogen bonding
• DNA ligase is added that joins foreign DNA to the plasmid
  
  • Sticky ends join permanently through covalent bonding

Question 29

Outline the process of electroporation

Answer 29

• Cells are briefly placed into an electric field to shock them
• Holes are created in their plasma membrane allowing plasmids to enter the bacterium

Question 30

Outline the process of inserting a plasmid into a bacterium (transforming)

Answer 30

• The bacterium is chilled in an ice bath
• It is then transferred to a 42°C environment, producing a heat shock
• Fluidity of the plasma membrane increases which increases chances for plasmid uptake

Question 31

State how selective markers are used to identify transformed bacteria

Answer 31

• Selective markers are genes that help identify transformed bacteria
• GFP (selective marker) makes transformed bacteria glow green under UV light
• Other examples include: ampicillin and insulin

Question 32

List the applications of gel electrophoresis

Answer 32

• Forensic investigations
• Paternity testing
• Identifying animals

Question 33

Give an example of how genetic modification can increase crop productivity

Answer 33

• Herbicide-tolerant GM canola can be sprayed with herbicide and only the weeds will die
• Weeds are controlled allowing for increased crop yield

Question 34

Give an example of how genetic modification can provide resistance to pests or disease

Answer 34

• Bt cotton (transgenic) has a bacterial gene that allows it to produce its own pest-resistant chemicals

Question 35

Outline the benefits associated with genetic modification

Answer 35

• Greater crop yield helps lower costs for both farmers and consumers
  
  • Many GMO plants have been modified to resist harmful environmental factos such as pests, floods and droughts
• Can also increase the nutritional value of food
  
  • E.g. Golden rice has high levels of beta-carotene (vitamin A)

Question 36

Outline the concerns associated with genetic modification

Answer 36

• Vegetarians may be concerned that animal genes are being used in GM plants
• Foreign genes in GMO foods may trigger allergic reactions

Question 37

Compare genetic modification to other methods

Answer 37

• Genetic modification is typically preferred when altering the genes of an organism
• This is due to its efficiency and accuracy
• It is commonly used in agriculture, but may also be used to modify living organisms to obtain a desired trait, such as the size of fish

Question 38

List the characteristics that make a plasmid good for recombination

Answer 38

• Have a variety of restriction sites
• Have an antibiotic resistant marker (selective marker)
• Have a promoter or origin point for self replication

NOTE: The ORI (labelled on a diagram) is the site at which the plasmid replicates.

Question 39

Outline how synthetic insulin is produced

Answer 39

• The A chain and the B chain are synthesised separately on separate plasmids in separate bacteria
• The plasmid used has antibiotic resistance selectable markers to allow for recombinant plasmid selection
• Before insertion into the plasmid, the insulin chain is modified toremove all introns(prokaryotes do not have introns)
• The insulin genes are insertednext to a gene for β-galactosidase protein, which allows fordetection of successful gene insertion
• Expression of each genefrom the two bacteria allows a functional fusion protein to be produced
• Once the genes are expressed and the fusion proteins are produced by each bacteria, these fusion proteins are then purified
• The insulin polypeptides are removed and thencombined together to produce functional insulin

NOTE: Human insulin is composed of 2 polypeptide chains; an A chain and a B chain (it is therefore a quaternary structure).

Question 40

State the function of the β-galactosidase (beta gal) protein

Answer 40

• Serves as an additional selective marker
• It is an inducible operon and therefore can regulate A chain production

Question 41

List the advantages of producing proteins through the expression of cloned genes as compared to the extraction from other biological sources

Answer 41

• High levels of purity
• Reliability of supply
• Reduced chance of side effects such as allergy (compared to pig or cow derived insulin)
• Consistency of quality between batches

Question 42

Explain why plasmids are referred to as vectors

Answer 42

• Vectors (in the context of gene editing technology) are agents that carry particular DNA segments into host cells
• Plasmids deliver a gene of interest into bacterial cells

Question 43

Outline how the gene for human insulin is cloned using gene cloning

Answer 43

• Target DNA is extracted from a cell or is made using reverse transcriptase
• Plasmids are cut with the same restriction enzyme (to create complementary sticky ends) and the gene for ampicillin/antibiotic resistance is added
• The plasmids, target DNA and DNA ligase are mixed to enable the plasmid to incorporate the target DNA
• Bacterial cells are then treated to make them permeable to the plasmids (through a heat shock/electroporation)
• The bacterial cells that have taken up the plasmid are identified (using the selective markers)

Question 44

State what can be done when no conclusions can be made from the results of gel electrophoresis

Answer 44

• Change the endonuclease being used
• Change the DNA section being tested

Question 45

Explain how gel electrophoresis can be used to determine if a plasmid is recombinant

Answer 45

• Adding foreign DNA to make a plasmid recombinant would increase its size
• When run through the gel electrophoresis, the banding pattern of the plasmid, control sample and the standard can be compared
• Additional bands in the plasmid sample that are not present in the control are the foreign DNA, indicating that the plasmid is recombinant