6.3 Manipulating Genomes Flashcards
DNA sequencing
technique that allows you to read the sequence of bases in dna
First method of dna sequencing
invented by Fred Sanger = Sanger method / chain termination methd
Explain the Sanger method
- He would get the dna double strand and turn it into a single strand
- He would allow the strands to be broken into diff bits of diff lengths
- He had 4 beakers, in each beaker he added that mixture
- In the first beaker he’d add the letter A but a modified version (radioactive dna) that whenever it’s incorporated the chain would stop and not extend further (second had modified c, 3rd = modified t, 4th = modified g)
- In the beaker there’s also normal nucleotides, dna polymerase and primers (short sequences of dna that act as the starting bit for dna polymerase to work = dna polymerase will start working to form complementary strands, each time a base would be added the chain would be stopped, thousands of chains of diff lengths and if u order them in terms of shortest to longest length you would get the sequence)
How to order the chains from the Sanger method
Use gel electrophoresis to find out shortest to longest
Small chains = further away after a set amount of time because longer chains would take longer to travel
Benefit of modified bases in Sanger method
modified in a way that they’re radioactively labelled which means during the gel electrophoresis process they’re easier to see.
How do they get lots of strands of the template for the Sanger method
- They get multiple strands of the template through PCR (way to amplify dna = polymerase chain reaction) or get your dna and put it into the plasmid of a bacteria = reproduce rlly fast
Can you sequence a whole genome
NO it would take too long
High throughput sequencing (HTS)
umbrella term for sequencing being automated + faster now
Example of HTS method
Pyrosequencing
What’s pyrosequencing
instead of using radioactively labelled dna, they took away the radioactivity = no longer radioactive but it was able to generate light so each letter basically had a diff light colour = each letter had a diff light intensity
How to work out which base is which in pyrosequencing
use mass spec to get peaks instead. = first peak was x length which shows its this base
What was pyrosequencing used for
Sequencing the human genome
Human genome project
diff scientists form diff parts of world sequenced genome + combined it all together
Why was the human genome project important
can now do genome wide comparisons between diff organisms = shows evolutionary relationships.
The human genome conserved genes
most conserved genes must have played an important role in the development of an organism
Methylation
our dnas can be methylated in different ways which can affect how 2 organisms are even though are genes are the same = affects how genes are turned on or off
What is methylation an example of
Epigenetics
What’s Epigenetics
factors that affect your dna without affecting your base sequence
What can you predict through genome sequencing
amino acid sequence of proteins
Synthetic biology
use your biological knowledge to create something artificial
Applications of synthetic biology
making proteins, biosensors, nanotechnology, medications
Bioethics
ethical issues of using synthetic biology
DNA profiling
- using dna you can create a profile and use this for CSI (forensics, paternity tests etc)
- Using dna technology to check each person has a specific profile
How to see banding pattern in dna profiling
- Through gel electrophoresis you can get a banding pattern of your dna which will be different to others
VNTRs?
Variable number of tandem repeats
What are VNTRs
sequences of dnas that are used in dna profiling. Every person has a different number of tandem repeats
What are tandem repeats
repetitive segments of dna that don’t code for a protein. They’re usually 10-100 base pair long
Outliner dna profiling
- 1 Obtain dna from mouth swabs, blood, hair, bone remains
- 2 Digest it w restriction enzymes = if you’re comparing 2 peoples dna you must ensure the same restriction enzyme is used
- 3 load it onto the gel electrophoresis
- 4 the smaller the fragment, the further it’ll travel
- 5 a banding pattern is produced
- 6 compare the banding pattern e.g. for paternity test the banding pattern of the child should be half of the mum’s and half of the dad’s
PCR?
Polymerase chain reaction
What’s pcr used for?
Used to amplify dna
What’s needed for PCR
Double stranded DNA - to act as a template
Free nucleotides (A,G,C,T)
DNA primers - signals to Taq polymerase where to bind and start synthesising
Taq polymerase - form of DNA polymerase (catalyses formation of H bonds between bases)
Buffer - maintains pH
Where does pcr ocur
In a thermocycler
Where’s taq polymerase found
Extracted from thermophilic bacteria
What is thermophilic bacteria
Can withstand high temperature without denaturing
Steps of pcr
Denaturing, annealing, elongation
Denaturing
happens at around 94-96 degrees
Denaturing happens so hydrogen bonds between complementary bases break.
What do you start pcr.
your dna you want to replicate and nucleotides and primers and dna taq polymerase + it’s co factor magnesium ions
Annealing
mixture cooled down to 68 degrees for this to happen. Primer binds to strand
Elongation
mixture heated to 72 degrees. DNA polymerase works forming phosphodiester bonds between nucleotides. ALWAYS 5’ TO 3’
What happens after the 3 steps of PCR
- Whole process repeats itself for many cycles to create more. Exponential growth (1 to 2, 2 to 4, 4 to 8)
Applications of PCR
- research e.g. if you wanna find a mutation
- Covid tests = identifying viral infection + perform as many tests as you want on the copies
What does electrophoresis do
- separate proteins or dna based on their sizes
Explain set up of gel electrophoresis
- You have a gel and make wells in it at the top and load it up with DNA.
- Put a power supply in the tank with a cathode on the side near the wells and the anode on the other side (dna is negative so it’ll move towards the anode)
How is the banding pattern created in gel electrophoresis
- Bigger DNA moves slower and smaller dna will move faster so it’ll be visible further from the well. = creates banding pattern
How to prepare dna for electrophoresis (GE)
creating fragments using restriction enzymes
Restriction enzymes
type of enzyme that can cut DNA at specific points.
If doing GE of child and parents dna what should u do when preparing
Child’s and parent’s dna should be cut w the same restriction enzyme so that they all form the same strand
What to add to dna in GE
loading dyes in the dna so it show up in the gel.
Why is GE sensitive
gel can’t be pierced it has to be stubbed very lightly
What can GE also be used for
Separating proteins
Explain GE for proteins
Proteins of diff sizes loaded into the wells and they’d be separated according to the wells
Example of GE for proteins
for haemoglobin bc in certain cases e.g. sickle cell the haemoglobin has a diff mass so you can compare normal haemoglobin to other haemoglobin to help w diagnosis.
When do u use dna probes
When identifying specific DNA sequences in samples
Commonly applied in genetic testing, disease diagnosis, and research
What is. DNA probe
complementary sequence to what you’re investigating) and the probe is usually fluorescently labelled.
What happens if the sequence is present
Probe binds to the dna
What are dna probes usually used for
Identifying a specific gene which can then be used for investigation e.g. diagnosis
What are microarrays used for
Investigating multiple diseases at once
Outline what a microarray looks like
- It’s a tray that has 100s of diff probes for diff things your investigating for = large scale of dna probes
Preparation for microarray
DNA must be broken into fragments
DNA labelled with fluorescent marker and there should always be positive and negative controls on the tray.
What are the steps involved in genetic engineering?
Obtain the required gene.
Place a copy of the gene inside a vector.
Carry the vector with the gene into a recipient cell
Method 1 for obtaining the required gene for genetic engineering
- obtain mRNA from cell where yk the gene is expressed
- use reverse transcriptase to obtain coding DNA sequence
How to make the dna become double stranded
add a primer (short sequence of dna that activates dna polymerase), nucleotides and dna polymerase.
Method 2 for obtaining the required gene for genetic engineering
If yk dna sequence of the gene - use automated polynucleotide synthesiser to make the gene
Multiply the one gene using pcr
Method 3 for obtaining the required gene for genetic engineering
- if yk person’s dna, use a probe to locate the desired gene and cut using restriction enzymes
Placing a copy of the gene inside a vector
cut the plasmid using restriction enzymes and use the same restriction enzymes to cut the dna.
When the gene is added it should be complementary
What is the vector usually for genetic engineering
plasmid = a bacteria’s dna
What happens when you cut the dna and plasmid with restriction enzymes
you get sticky ends = unpaired nucleotide bases
How do you anneal the plasmid in genetic engineering
Using DNA ligase enzyme
Method 1 for the vector carrying the gene into a recipient cell
heat shock treatment (dip bacteria into really cold ice and then heating it up really fast) if you keep going from cold to hot to cold to hot and use calcium chloride, the phospholipid membrane becomes porous and the plasmid can enter the bacteria
Method 2 for the vector carrying the gene into a recipient cell
electroporation = using a high voltage to disrupt the cell membrane so that the plasmid can go into the bacteria
Method 3 for the vector carrying the gene into a recipient cell
electrofusion = electrical current is used to introduce the dna into the bacteria. Electrofusion doesn’t damage membrane but electroporation does.
Method 4 for the vector carrying the gene into a recipient cell
transfection = there are viruses that can affect bacteria’s called bacteriophages which can transfect the host cell = load up the plasmid into those viruses and the viruses will then infect the bacteria.
How to directly introduce a gene into a recipient
- if tumour bacteria doesn’t work inside of plants
- use gene gun
What’s a gene gun
device where you cost the dna in something stable like gold and then shoot it into the plant
Where is reverse transcriptase found and what is it
retroviruses, such as HIV, have an RNA genome which contained the enzyme reverse transcriptase
Purpose of reverse transcriptase
- changes RNA into being seen as DNA
- when HIV infects a cell, it will think that HIV’s instructions are it’s own so starts following those instructions + making more HIV
What are restriction enzymes
can make cuts that provide sticky ends or they can have blunt cuts w no sticky ends
What’s DNA ligase function
Seals dna
Source of insulin mRMA
Beta cells of the islets of langerhans
Steps for genetically modifying bacteria to have insulin
- use reverse transcriptase enzyme to convert mRNA into complementary DNA = obtain gene for insulin
- add unpaired nucleotides to ends to create sticky ends
- use ligase enzyme to put the insulin gene in the plasmid
- use heat shock to put the plasmid into the bacteria
- test of bacteria has incorporated plasmid using replication plates
How to test the bacteria had incorporated the plasmid
- bacteria usually resistant to two different types of antibiotics but insulin gene interferes with one of the antibiotic resistance genes.
Test this out using agar plates using agar plates w the antibiotics on them. First use the agar plate w the antibiotic yk it should be resistant to and then use the agar plate w the antibiotic it shouldn’t be resistant to
Ethical issue of genetic modification (GM) - escaping
- If you’re using a bacteria and you’re modifying it to make it antibiotic resistant, that can escape into the wild = to ensure this they have other genes knocked out of them to prevent them from synthesising the nutrients they need to survive in the wild
Ethical issue of GM - plants
- Plants = have been modified to produce toxins so they can be pesticide resistant but this is toxic to other organisms such as butterflies.
Ethical issues of GM - soya beans
made them resistant to herbicides = problem is this resistant gene may be able to spread into weeds and become herbicide resistant weeds
Ethical issues of GM (golden rice)
GM rice which contains a gene to produce beta carotene = needed to make vitamin a in the body. Ethical concern = not all farmers would have access to it so expensive to some and those farmers would lose their business but company that came up w golden rice said everyone in India gets free license to grow it
Ethical issues of GM (resistance)
- Crop plants that are pesticide resistant= some people don’t want GM products
Ethical issues of GM (vaccines)
- Viruses can be genetically modified for medical purposes = can be used as vaccines by taking away the protein that makes them harmful. Viruses used in cancer treatment if modified = can increase the risk of cancer if start creating cuts in normal dna = mutation
Ethical issues of GM (mice)
- Mice can be genetically modified = can study using them. Ethical concern = animal testing = strict protocols to perform animal testing without it being unethical
Ethical issues of GM (silk)
- silk = genetically modified goats to produce the spider silk protein in their milk. = ethical concern = welfare of goat
Electroporation
way of inserting the plasmid into the bacteria
2 types of gene therapy
Germ line therapy
Somatic cell gene therapy
Somatic gene cell therapy
modifying a body cell = nothing should be passed onto offspring
Germ line therapy
change genome of the gametes or zygotes which means individuals genome would be altered + the offspring may also inherit the modified alleles
Gene therapy
treats diseases that are caused by a problem w the gene (mutated or non functional) e.g, cystic fibrosis
- changes alleles to correct the genetic issue
Genetic issue
too much or too little of something is made. If too much is made then translation in that cell can be blocked.
Cystic fibrosis
homozygous recessive disease which affects the CFTR allele (type of protein channel) = means when mutated there’s an abnormal allele = lack of expression of that protein. This means that they have abnormal chloride ion channels.
How to treat cystic fibrosis
Would want to give epithelial cells alleles to make chloride ion channels
How would you give epithelial cells the alleles to make chloride ion channels
- take liposomes (fat droplets) and inserting the functioning allele inside
Liposomes would diffuse straight through the plasma membrane and pass through the nuclear envelope and the allele would be inserted into the host’s genome. It’s as if the host has been virally infected so they will start making CFTR proteins (the functioning version) as a response.
How do you take liposomes and insert the functioning allele inside
Done through aerosols = sprayed through the nose = will go to the epithelial cells of the respiratory tract
Problem w current treatment of cystic fibrosis
this isn’t a cure, it’s a treatment. Because the epithelial cells get replaced in the lungs every two weeks, this treatment would have to be continuously provided
What is the treatment of cystic fibrosis an example of
Somatic gene cell therapy
What else can be used as a vector just like liposomes
Viruses
What type of gene therapy is using viruses as a vector like liposomes
Somatic gene cell therapy
Why’s it dangerous to use viruses as a vector just like liposomes
- Even though the virus isn’t supposed to be virulent (active) it can still provoke an immune response.
- The patient may become immune to the virus so the same virus can’t be used as a vector again.
- Virus may insert the dna into the wrong place in the genome = increasing the risk of cancer
Germ lime gene cell therapy
editing the genome of a gamete or zygote. Because the gamete or zygote is being edited the allele can be passed onto offspring.
Why is germ line gene cell therapy unethical
Offspring never consented to this
Other issues w germ line gene cell therapy
concerns on safety of how they’d insert genes into the gamete or zygote = if done wrong it could kill gamete/zygote or give rise to cancers. Due to ethical concerns this is illegal.
Epidemiology
branch of medicine where you study the spread and occurrence of diseases. = can be researched better and figure out epidemiology of a virus by looking at their genes
