chapter 3&4 (enzymes & DNA manipulation) Flashcards
endonucleases
- enzymes responsible for cutting DNA strands
- cleaves the phosphodiester bonds
BLUNT: no overhanging nucleotides
STICKY: overhanging, unpaired nucleotides
linear v circular DNA fragments
LINEAR:
2 recognition sites and 3 fragments
CIRCULAR:
2 recognition sites and 2 fragments
ligases
- 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
polymerases
- add nucleotides
- amplify sections of DNA or RNA
- primers are required to attach polymerases
CRISPR-Cas9 in bacteria
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
CRISPR-Cas9 in gene editing
- 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
limitations of CRISPR-Cas9 (gene editing)
- 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
What is polymerase chain reaction (PCR)
- 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)
PCR steps
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
forward and reverse primers
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
What is gel electrophoresis?
- used to measure size of DNA fragment after DNA has been cut up
Steps for gel electrophoresis
- DNA samples placed in wells
- standard DNA fragments with known sizes are loaded
- gel is made of agrose
- gel is immersed in buffer solution - Electric current is passed through the gel using two electrodes, one positive and one negative
- negative electrode is the well end - Smaller DNA fragments move further and faster through gel
- they are now separated based on size - gel is stained with fluorescent dye and visualised under UV light
gel electrophoresis interpreting gels, genetic testing & crime scene use
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
making recombinant plasmids
- 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
gene of interest
- sequence of DNA encoding the protein we wish to generate
- DNA sequence is amplified using PCR before it is inserted into a vector
plasmid vector
- 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)
uptake of recombinant plasmids: heat shock
- 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
uptake of recombinant plasmids: electroporation
- 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
transformed bacteria and anti-biotic selection
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
protein production & extraction
- transformed bacteria are cultured and induced to produced target proteins
- protein of interest is extracted and purified
insulin
- responsible for regulating blood glucose levels
- insulin can be produced by transformed bacteria
- insulin has a quaternary structure consisting of 2 polypeptide chains
insulin steps
- plasmids vectors are prepared to encode antibiotic resistance
- endonuclease cuts both genes to form sticky ends
- plasmids are added to E.coli bacteria solution and undergo either heat shock or electroporation to increase uptake
- bacteria is spread and incubated
- recombinant plasmids will produce insulin subunit
- transformed bacteria is placed into conditions to reproduced
- two insulin chains are mixed together which allows bonds to form and create functional insulin
genetically modified organisms
- alterations of an organisms genome
- the organism that receives the altered gene is the host organism
transgenic organisms
- genes from different species inserted into it’s genome
- means it is able to produce proteins not previously part of their species
cisgenic organisms
- genes from same species inserted into it’s genome
How GMOs are used in agriculture
- increase crop productivity (quality, nutrition, amount of food)
- increase disease resistance of the crop (crop loss from pathogens and pests)
Producing transgenic plants
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)
golden rice pros and cons
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
Issues surrounding GMOs:
BIOLOGICAL
BIOLOGICAL PROS:
- better crop productivity
- insect resistant
- improved nutritional content
BIOLOGICAL CONS:
- crops may lose effectiveness
- loss in genetic diversity
- cross-pollination
Issues surrounding GMOs:
SOCIAL
SOCIAL PROS:
- better food security
- larger profits for farmers
- improve customer appeal
SOCIAL CONS:
- expensive
- complex legal issues
Issues surrounding GMOs:
ETHICAL
ETHICAL PROS:
- wide spread benefits
- overall health of humanity
ETHICAL CONS:
- unnatural (playing god)
- unsafe to eat
- modifying animals = inhumane
features of enzymes
- 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
activation energy
- enzymes lower activation energy
- all reactions have activation energy
- getting the reactants to the products requires less energy with an enzyme
anabolic & catabolic
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)
what does the enzyme reaction look like
- substrate enters the active site
- creates enzyme-substrate complex
- the reaction occurs
- products of reaction the leave then active site of the enzyme
