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Biology 3/4 > chapter 3&4 (enzymes & DNA manipulation) > Flashcards

chapter 3&4 (enzymes & DNA manipulation) Flashcards

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1
Q

endonucleases

A
  • enzymes responsible for cutting DNA strands
  • cleaves the phosphodiester bonds
    BLUNT: no overhanging nucleotides
    STICKY: overhanging, unpaired nucleotides
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2
Q

linear v circular DNA fragments

A

LINEAR:
2 recognition sites and 3 fragments
CIRCULAR:
2 recognition sites and 2 fragments

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3
Q

ligases

A
  • enzyme that joins two fragments
  • formation of phosphodiester bonds
  • can join sticky or blunt ends
  • sticky ends are better due to making more bonds when ‘glued’ together
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4
Q

polymerases

A
  • add nucleotides
  • amplify sections of DNA or RNA
  • primers are required to attach polymerases
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5
Q

CRISPR-Cas9 in bacteria

A

EXPOSURE:
- the bacteriophage injects in DNA
- a short section of the viral DNA is cut out called the protospacer
- the protospacer is introduced into the bacterium CRISPR gene becoming a spacer
EXPRESSION:
- CRISPR spacers are transcribed along with half a palindrome and converted into gRNA
- gRNA binds to Cas9 to create CRISPR-Cas9 which is directed to any viral DNA
EXTERMINATION:
- the CRISPR-Cas9 scans for invading bacteriophage
- Cas9 cleaves the phosphate-sugar backbone and creates blunt ends

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6
Q

CRISPR-Cas9 in gene editing

A
  • induce genetic change by cutting DNA at a specific location chosen
    (added or removed nucleotides and change the function of a gene)

STEPS:
- synthetic gRNA is created that matches target DNA
- Cas9 enzyme is obtained with target PAM sequence
- Cas9 and gRNA bind together to make the CRISPR-Cas9 complex
- mixture is injected into specific cells
- Cas9 finds target PAM sequence
- Cas9 cuts the selected sequence
- DNA with blunt end will attempt repair
- when repairing cells may introduce new nucleotides

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7
Q

limitations of CRISPR-Cas9 (gene editing)

A
  • it can be difficult to achieve and is not consistently successful
  • illegal to modify human embryos and put them into women
    SAFTEY: off target cleavages
    INFORMED-CONSET: embryos cant consent
    INEQUAILITY: only wealthy (expensive)
    DISCRIMINATION: judged by society
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8
Q

What is polymerase chain reaction (PCR)

A
  • amplifies sample of DNA
  • is a DNA manipulation technique which makes multiple copies of DNA
  • every number of cycles it doubles
    (0 cycles = 1, 1 cycle = 2, 2 cycles = 4)
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9
Q

PCR steps

A

DENATURATION
- DNA is heated to 90-95 degrees to break hydrogen bonds, forming single stranded DNA
ANNEALING
- DNA is cooled to 50-55 degrees to allow primers to bind to complementary sequences
ELONGATIONS
- DNA is heated to 72 degrees, allowing Taq polymerase to bind to the primer and being synthesising
REPEAT

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10
Q

forward and reverse primers

A

FORWARD:
- will bind to the start codon at the 3’ end of the template strand, this causes Taq polymerase to synthesise a new DNA strand in the same direction
REVERSE:
- will bind to the stop codon at the 3’ end of the coding strand, this causes Taq polymerase to synthesise a new DNA strand in the reverse direction

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11
Q

What is gel electrophoresis?

A
  • used to measure size of DNA fragment after DNA has been cut up
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12
Q

Steps for gel electrophoresis

A
  1. DNA samples placed in wells
    - standard DNA fragments with known sizes are loaded
    - gel is made of agrose
    - gel is immersed in buffer solution
  2. Electric current is passed through the gel using two electrodes, one positive and one negative
    - negative electrode is the well end
  3. Smaller DNA fragments move further and faster through gel
    - they are now separated based on size
  4. gel is stained with fluorescent dye and visualised under UV light
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13
Q

gel electrophoresis interpreting gels, genetic testing & crime scene use

A

INTERPRETING GELS
- molecular size indicates length of nucleic acid sequence
- compared with other bands we can estimate molecular size
- thicker band = contains more DNA
GENETIC TESTING
- first undergoes PCR
- having a standard ladder (to help identify size), healthy gene, mutated gene and the individuals sample
CRIME SCENE
- extract DNA
- undergoes PCR
- using DNA profiling we can discover how related people are and compared suspect DNA
- heterozygous = two bands
- homozygous = one thick band

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14
Q

making recombinant plasmids

A
  • insert foreign DNA into a plasmid that can be taken up by bacteria
  • bacteria will the express the protein
    TO CREATE THEY REQUIRE:
  • gene of interest
  • a plasmid vector
  • restriction endonuclease
  • ligase
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15
Q

gene of interest

A
  • sequence of DNA encoding the protein we wish to generate
  • DNA sequence is amplified using PCR before it is inserted into a vector
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16
Q

plasmid vector

A
  • where gene of interest will be inserted
    a plasmid must contain two genes that code for observable traits antibiotic resistance gene and reporter gene (identifies whether plasmid has accepted gene of interest)
17
Q

uptake of recombinant plasmids: heat shock

A
  • bacteria and plasmids are placed in solution on ice
  • then placed in solution that is heated to approx 37-42 degrees for 25-25 seconds
  • causing plasma membrane to become more permeable and allows plasmid vectors to cross phospholipid bilayer
18
Q

uptake of recombinant plasmids: electroporation

A
  • instead of heat electric current is added to the plasmids and bacteria
  • causing plasma membrane to become more permeable and allows plasmid vectors to cross phospholipid bilayer
19
Q

transformed bacteria and anti-biotic selection

A

bacterial transformation is bacteria with recombinant plasmids
- only transformed bacteria will have the gene necessary for anti-biotic resistance, all other untransformed will be killed off
visible colonies show on petri-dish
= transformed bacteria

20
Q

protein production & extraction

A
  • transformed bacteria are cultured and induced to produced target proteins
  • protein of interest is extracted and purified
21
Q

insulin

A
  • responsible for regulating blood glucose levels
  • insulin can be produced by transformed bacteria
  • insulin has a quaternary structure consisting of 2 polypeptide chains
22
Q

insulin steps

A
  1. plasmids vectors are prepared to encode antibiotic resistance
  2. endonuclease cuts both genes to form sticky ends
  3. plasmids are added to E.coli bacteria solution and undergo either heat shock or electroporation to increase uptake
  4. bacteria is spread and incubated
  5. recombinant plasmids will produce insulin subunit
  6. transformed bacteria is placed into conditions to reproduced
  7. two insulin chains are mixed together which allows bonds to form and create functional insulin
23
Q

genetically modified organisms

A
  • alterations of an organisms genome
  • the organism that receives the altered gene is the host organism
24
Q

transgenic organisms

A
  • genes from different species inserted into it’s genome
  • means it is able to produce proteins not previously part of their species
25
Q

cisgenic organisms

A
  • genes from same species inserted into it’s genome
26
Q

How GMOs are used in agriculture

A
  • increase crop productivity (quality, nutrition, amount of food)
  • increase disease resistance of the crop (crop loss from pathogens and pests)
27
Q

Producing transgenic plants

A

GENE IDENTIFICATION:
- gene of interest must be identified and isolated
GENE DELIVERY:
- delivered into the cells of the host (via direct insertion or through plasmids)
GENE EXPRESSION:
- transformed cells then grow repeatedly
(host organism now expresses the new transgene)

28
Q

golden rice pros and cons

A

PROS:
- increases beta-carotene = increase in vitamin A
- kept and re-planted
- fewer deaths
CONS:
- reduce crop biodiversity
- might interfere with previous vitamin A supplements

29
Q

Issues surrounding GMOs:
BIOLOGICAL

A

BIOLOGICAL PROS:
- better crop productivity
- insect resistant
- improved nutritional content
BIOLOGICAL CONS:
- crops may lose effectiveness
- loss in genetic diversity
- cross-pollination

30
Q

Issues surrounding GMOs:
SOCIAL

A

SOCIAL PROS:
- better food security
- larger profits for farmers
- improve customer appeal
SOCIAL CONS:
- expensive
- complex legal issues

31
Q

Issues surrounding GMOs:
ETHICAL

A

ETHICAL PROS:
- wide spread benefits
- overall health of humanity
ETHICAL CONS:
- unnatural (playing god)
- unsafe to eat
- modifying animals = inhumane

32
Q

features of enzymes

A
  • reusable
  • specific
  • reversible (work in both directions)
  • speed up, not create
  • have an active site
  • are proteins
  • are a subset of catalysts
  • act on entire biochemical pathways
  • end in ‘ase’
  • above the arrow
33
Q

activation energy

A
  • enzymes lower activation energy
  • all reactions have activation energy
  • getting the reactants to the products requires less energy with an enzyme
34
Q

anabolic & catabolic

A

anabolic: two or more molecules combine to form larger one (e.g building things up)
catabolic: larger molecules turning into two or more smaller ones (e.g breaking things down)

35
Q

what does the enzyme reaction look like

A
  • substrate enters the active site
  • creates enzyme-substrate complex
  • the reaction occurs
  • products of reaction the leave then active site of the enzyme

Biology 3/4 (10 decks)

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