Chapter 2: DNA manipulation

Question 1

DNA polymerase

Answer 1

uses DNA polymerase to accurately copy a DNA template

Question 2

endonucleases - restriction enzymes

Answer 2

• restriction enzymes like endonucleases cut DNA at specific recognition sequences known as restriction sites, splitting DNA into smaller fragments
  
  • restriction sites is a particular order of nucleotides
• endonucleases make one incision on each of the 2 complementary strands of DNA

Question 3

Sticky ends

Answer 3

• endonucleases cut one strand at one point but cut the second strand at a point that is not directly opposite → causes an overhang to form
• DNA ligase enzyme connects the single-stranded DNA together via the sugar-phosphate back bones

Question 4

which pairs faster: sticky or blunt

Answer 4

• pieces of DNA with sticky ends pair faster than pieces of DNA with blunt ends
  
  • sticky ends allow for complementary base pairing so the pieces are held together by weak hydrogen bonds and can be acted on by the DNA ligase enzyme.

Question 5

Blunt ends

Answer 5

• endonuclease cuts the 2 strands of DNA molecule at points DIRECTLY OPPOSITE each other to produce cut end
• DNA fragments are joined directly together through the use of DNA ligase

Question 6

DNA ligase

Answer 6

an enzyme known as ligases catalyses the joining of pieces of double-stranded DNA at their sugar-phosphate bones

Question 7

What are plasmids

Answer 7

• circular, double stranded DNA that can reproduce independently
  can be taken up by bacterial cells
  
  • many are used as vectors to transport foreign DNA into bacterial cells to transform them
  • they have multiple recognition sites (restriction sites) for endonucleases
  • can have new genes inserted
• have an antibiotic resistant marker (selective marker)
• have a promoter or origin point for self replication

Question 8

Making recombinant plasmids

Answer 8

• cut plasmid DNA with endonuclease
• cut foreign DNA with the same endonuclease
• mix plasmids and foreign DNA
• add DNA ligase which joins the sticky ends together
• same recognition site allows same restriction enzyme to cut both the plasmid and the gene of interest
• produces matching sticky ends on the plasmids and the gene of interest
• plasmids will then join with the DNA of the gene of interest to form recombinant plasmids as they have complementary sticky ends.
• the two fragments can then be successfully joined by DNA ligase.

Question 9

recombinant plasmid

Answer 9

a plasmid that has taken in new DNA is called a recombinant plasmid

Question 10

bacterial transformation

Answer 10

• when bacteria cells take up plasmids
• recombinant plasmids need to be taken up by bacteria
• techniques that temporarily interfere with the plasma membrane allow this (causes holes in the pm so plasmid can enter)
  
  • electroporation
  • heat shock

Question 11

how is transformed bacteria identified

Answer 11

• bacteria can take up recombinant plasmid with AmpR gene
  
  • results in bacteria resistant to ampicillin antibiotic
• bacteria can take up recombinant plasmid with green fluorescent protein (gfp gene)
  
  • when uv light is used, bacteria is fluorescent
  • but needs arabinose , sugar to repress the repressor of the gfp gene

Question 12

ethical issues of gene cloning

Answer 12

• changing a species’ DNA may result in unforeseen consequences
• concern that the pharmaceutical product may contain bacteria that will cause disease
• not natural and therefore may be against religious or moral views
• 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

purpose of LB broth

Answer 13

• provides nutrients to support bacterial growth
• both transformed and untransformed bacteria can grow

Question 14

purpose of ampicillin

Answer 14

• to identify which bacteria has been transformed
• bacterial growth → has the +pREC because it has AmpR gene, resistant to ampicillin antibiotic
• no bacterial growth → is -pREC no AmpR gene

Question 15

purpose of arabinose

Answer 15

• to also identify which bacteria has been transformed
• arabinose represses the repressor of the GFP gene (binds to protein AraC)
  allow the GFP gene to be expressed
• so under UV light, bacteria will appear green

Question 16

potential errors
- transforming bacteria experiment

Answer 16

• bacteria was left in the heat bath for too long, killing the bacteria before plating it
• the wrong test tube was put on the plate
• no recomb test tube was added to the wrong plate (plates mixed up) → human error

Question 17

benefits of recombinant insulin

Answer 17

• high levels of purity
• reiability of supply
• reduced chance of side effects such as allergies (compared to pig or cow derived insulin)
• consistency of quality between batches

Question 18

gel electrophoresis

Answer 18

• a method used to separate DNA fragments based on size (length, measured in bp)
• the phosphate group of nucleotides is negatively charged
• negatively charged DNA moved towards the positive terminal (b/c it is negatively charged)
• DNA is loaded into a gel that acts like a sieve to allow SMALLER DNA fragments through more quickly than larger ones
• the result of gel electrophoresis is a series of PARALLEL BANDS of DNA fragments at differing distances down the gel
• each band can contain millions of DNA molecules of the same size

Question 19

mitochondrial DNA

Answer 19

• not all DNA is used in gel electrophoresis
  
  • nuclear or mitochondrial DNA can be used
• compared to nuclear DNA, mtDNA
  
  • is inherited via the MATERNAL LINE
  • does not recombine during reproduction → LESS VARIABLE
  • is present in LARGER AMOUNTS

Question 20

applications of gel electrophoresis

Answer 20

• people have DIFFERENT DNA SEQUENCES, so when endonucleases are used, the DNA will be cut at DIFFERENT LOCATIONS and DNA fragments of different lengths will result

gel electrophoresis can identify people in:
- forensic investigations
- mass disasters
- paternity testing
- identifying animals

Question 21

dna profiling - short tandem repeats

Answer 21

• chromosomal sites (non coding region) where many copies of a short DNA sequence are joined end-to-end; the number of repeats is variable between unrelated people
• STRs are 2-5 bp in length and repeated over and over
• the number of REPEATS VARIES between people and each variation is a distinct allele
• we have 2 copies of each STR - one from mother and one from father

Question 22

types of DNA used for DNA profiles

Answer 22

• to develop DNA fingerprints or DNA profiles, regions with high variation between individuals are used.
• Short Tandem Repeats (STRs) from nuclear DNA
  
  • STRs can identify individuals
• Hypervariable regions (HVRs) in mtDNA
  
  • mtDNA can identify relationships/when fewer cells are available

Question 23

how to run a gel electrophoresis

Answer 23

1. DNA sample is combined with DNA loading dye.
2. the mixture is placed in a well at one end of the agarose gel.
3. the agarose gel is immersed in a buffer solution (salt solution).
4. it is then exposed to an electric field with the positive(+)pole at the far end and the negative(−) pole (cathode) at the well
5. smaller fragments move through the agarose gel faster compared to larger fragments.
6. these fragments appear as bands on a gel which can be interpreted in various ways. This usually needs to be observed under UV light.

Question 24

what is more useful for gel electrophoresis- sticky or blunt ends

Answer 24

• blunt ends
• sticky ends may allow for the hydrogen bonds between the complementary base pairs to reform
• causes fewer bands on the gel - less reliable results

Question 25

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

Answer 25

• Adding foreign DNA to make a plasmid recombinant would increase its size.
• when run through the gel electrophoresis you can then compare the banding pattern of the plasmid sample to the control sample and DNA ladder of known sizes
• If you see additional bands in the plasmid sample that are not present in the control, these bands correspond to the insert DNA, indicating that the plasmid is recombinant.

Question 26

making synthetic insulin

Answer 26

• human insulin is composed of 2 polypeptide chains
  
  • an A chain and a B chain. It is therefore a quaternary structure.
• the A chain and the B chain are synthesised separately on separate plasmids in separate bacteria.
• Restriction endonucleases are used to cut, e.g. the plasmid or human DNA.
• DNA ligase joins the DNA sequences into the plasmid.
• the plasmid used has antibiotic resistance selectable markers to allow for recombinant plasmid selection.
• the insulin genes are inserted next to a gene for β-galactosidase protein, which allows for detection of successful gene insertion.
• The beta gal gene has 2 functions
  
  • its an additional selectable marker
  • the beta gal gene is an inducible operon and therefore can regulate A chain production
• processing of the protein such as joining insulin polypeptide chains A and B occurs to create functional insulin.