19: Gene Tech Flashcards
Why is having a small plasmid useful?
can be easily taken up by the cell
What property allows plasmids to be cut up?
has sites for restriction endonuclease
Why is independent replication for plasmids good?
allows for large amount of DNA to be transcribed/high copy rate of plasmids
`Why are plasmids good vectors?
5 reasons
- small - can be easily taken up by the cell
- sites for restriction endonucleases - can be cut up and new genes can be inserted
- circular - makes them stable
- marker genes - recombinant bacteria can be recognised
- replicate independently - allows for a large amount/high copy rate of plasmids
What is an inducible enzyme?
An enzyme that is only produced when the substrate it acts upon is present (the gene can be turned on or off, and the substrate controls that gene expression)
What are the benefits of bioinformatics?
- databases of DNA from all over the world
- analyse genetic information
- can compare between two samples of DNA
- identify genes
- predict the primary, secondary and tertiary structure of proteins
Why do you need to add a promoter when inserting an eukaryotic gene into a prokaryote?
Eukaryotic DNA does not have a promoter; gene expression is controlled by transcription factors instead. Prokaryotes do have promoters, and their RNA polymerase only binds to promoters, so in order for transcription to occur, a promoter needs to be added.
What are genes that have constituative expression and why are they needed?
They are genes that are always turned on, so transcription of that gene is continously happening, and the product is always made. This is needed because the proteins they produced are needed all the time.
In a microarray, what is dyed?
cDNA
In a microarray, what are the probes?
single stranded DNA (ssDNA)
Why do you wash the microarray with water?
To remove any cDNA that did not stick/bind to the ssDNA
How does the repressor protein affect gene expression in the lac operon?
Repressor protein binds to the promoter and prevents transcription. If lactose is present, the repressor protein does not bind to the promoter, it binds to the lactose, meaning that the RNA polymerase can then bind to the promoter.
What are the benefits of prenatal screening?
- termination of preganacy
- early treatment/warning (e.g. if CF is present)
- stop anxieties
What are the drawbacks of prenatal screening?
- increases the risk of miscarriage
What are the benefits of GMO?
- increases food sources
- increase nutritional value - decreases malnutrition
- increases wealth/economy
- resistant to herbicides/insecticides; increased yield and makes the most of the land available
What are the drawbacks of GMO?
- increased chance of ‘superweeds’ - resistant strains which if leaked to wild, could out-compete wild strains
- long term health impacts unknown
- pollen could transfer the resisitive gene to wild plant and produce hybrid offspring that are invasive weeds
- reduced genetic diversity; the species is more vulnerable to environmental changes
- need to buy new GMO seeds each year; expensive
Why does gel electrophoresis work?
DNA is negatively charged, so will move through the gel from negative terminal to the positive one
What are the benefits of embryonic screening?
- only implant highest quality embryos; embryos with no genetic disorders are only implanted (decreases prevalency of haemophilia, huntington’s etc.)
- decreases risk of failed pregnancy/miscarriage from embryos that wouldn’t survive
What are the drawbacks of embryonic screening?
- embryos thrown away - murder?
- pick and choose the qualities of the baby - designer babies (e.g. embryos thrown away because it is not the sex they wanted)
- expensive - funds better spent elsewhere
What are the 3 stages of PCR?
- denaturation
- annealing
- extension
What are the 3 temperatures of PCR, in order?
- 95
- 60
- 72
What is the name of the enzyme that makes cDNA from mRNA?
reverse transcriptase
What kind of DNA polymerase is used in PCR?
Taq polymerase from thermophilic bacteria: Thermus aquaticus
What does the lacZ gene code for?
lactase
What does the lacA gene code for?
acetyl transferase
What does the lacY gene code for?
permeate
What does lacI gene code for?
repressor protein
What kind of genes are lacZ, lacY and lacA?
Structural genes
What kind of gene is lacI?
Regulatory gene
What is a structural gene?
A gene that codes for a protein which has a function within a cell.
What is a regulatory gene?
A gene that codes for a protein which has a role in gene expression (the repressor protein).
What is a promoter?
Found in prokaryotes; a non-coding section of DNA (intron) that controls an operon. RNA polymerase binds here.
What is an operon?
A cluster of structural and regulatory genes, controlled by a single promoter.
Why are lacZ, lacY and lacA all transcribed at the same time?
They are controlled by the same promoter/in same operon.
What is DELLA?
A repressor protein, produced from a regulatory gene.
What is gibberellin?
A hormone - the determining molecule in weather transcription occurs.
What is PIF?
A transcription factor
What is a transcription factor?
Controls gene expression in eukaryotes. It is an enabler, it helps the RNA polymerase to bind.
Why can mRNA be used in a microarray to make cDNA?
If a gene is expressed, it must have been transcribed, so the corresponding mRNA must be present in the cytoplasm. If mRNA found in the cytoplasm is used and made into cDNA, if it then binds to ssDNA then we know that gene is present and is being expressed. mRNA is an indicator of the gene. It is also useful because it has no introns.
What is the purpose of a microarray?
To compare 2 tissue samples, to see which genes are expressed? None, one, or both?
What needs to be added to mRNA to make cDNA and why?
Add a poly-A tail (AAA) post-transcription and a RNA primer (UUU). This allows for reverse transcriptase to attach to the mRNA and start making cDNA.
What does the reverse transcriptase use to created the cDNA?
free DNA nucleotides
How can double stranded DNA be made from cDNA?
- add RNA-ase to digest the mRNA template
- add DNA polymerase, nucleotides and dNTPs
What are the 3 methods of synthesising DNA?
- using reverse transcriptase and mRNA
- artificial synthesis using genetic code (choosing codons for the amino acid sequence desired and joining them together)
- using restriction endonuclease to isolate the gene
What is recombinant DNA?
DNA made by artificially joining together pieces of DNA from 2 species.
What is a transgenic organism?
Any organism that contains DNA from another source.
What is a GMO?
Any organism that has had its DNA changed in a non-natural way.
What are three kinds of vector?
Plasmids, viruses, liposomes
What is the name of the enzyme which joins recombinant DNA together?
DNA ligase
Why might you need electric current when making a transgenic organism?
So the cell uptakes the plasmid.
What is a palindromic recognition sequence?
A pattern of bases on DNA or RNA which reads the same 5’ to 3’ as 3’ to 5’. Restriction endonuclease enzymes have specific palindromic sequences where they cut the DNA/RNA at.
How can plasmids be obtained?
Treat bacteria with enzymes to break down their cell walls and then spin the ‘naked’ bacteria in a centrifuge.
Advantages to recombinant medicines (insulin, factor VIII, adenosine deaminase)?
- less chance of rejection due to being human insulin and not an animal
- less ethical issues because not having to use other organisms as a source
- easier to administer, via injection, instead of blood transfusions
- can be made in larger quantities, very quickly
- not much space is needed
- can be carried out anywhere in the world
What is crispr?
a group of base sequences that code for short lengths of RNA that direct a endonuclease enzyme known as Cas9 towards specific base sequences. The endonuclease enzyme then cuts at that specific point.
What is the purpose of crispr?
To make sure that a gene is being inserted in the correct place and that the place is known. Basically to control where the gene gets inserted in the DNA.
What is gDNA and what is it made of?
Another name for the short lengths of RNA in crispr that direct an endonuclease enzyme to the correct location to cut. It is made of a sequence of 20 bases that binds to the complementary strand on the DNA.
Why is crispr/gDNA useful in gene editing?
gDNA can be made to be complementary to any base sequence of DNA. So DNA can be edited at any location along a genome.
Why does Cas9 have two active sites?
To cut both strands of DNA/break the sugar-phosphate backbone of both strands.
What is Cas9?
the restriction endonuclease associated with crispr, and is directed by gDNA to the correct location on the genome.
What can you do in gene editing?
‘silence’ a gene or ‘repair’/’replace’ a faulty allele by adding in 1+ nucleotides in.
What do dNTPs do?
provide the energy to synthesise a new sugar-phosphate backbone/phosphodiester bonds
What does the denaturation stage of PCR do?
breaks the hydrogen bonds between the 2 DNA strands. This exposes the bases.
What happens in the annealing stage?
Add primers (short lengths of ssDNA) so the DNA polymerase can attach.
Why do primers have to be added in PCR?
For the DNA polymerase to have something to attach onto
What happens in the extension stage of PCR?
the DNA polymerase synthesises a new DNA strand, using dNTPs
After PCR is completed, what happens?
The mixture is reheated to 95 to begin the second cycle. This separates the template strand and the new strand, so both can be copied.
What are the 3 examples of recombinant medications?
- insulin
- factor VIII
- adenosine deaminase
Pros to adult genetic screening?
- can make informed medical decisions in future i.e. pregnancy w/ Huntingtons
- stop anxieties
- make lifestyle changes/preventative measures
- earlier treatment (e.g. breast cancer - remove breasts, or start on medication )
- decide to go into clinical trials
- economic benefits because less money spent on long-term treatments for chronic conditions if caught early on
Cons to adult genetic screening?
- not everyone is eligible
- expensive
- emotionally draining (e.g. with Huntingtons if you test positive but have already had children, then they will 100% have it too).
- if you test for a recessive allele you have the stress of not knowing if the disease will develop of not
What is gene therapy?
The treatment of a genetic disorder by inserting genetically coreected cells into the body or introducing functional genes into affected cells.
What is the process of inserting functional genes into affected cells work in gene therapy?
A viral vector with the functional gene is inserted into the human cell with defective gene via endocytosis. This then embeds the viral DNA into the cells genome, which gets transcribed as normal into the functional protein.
Why are viruses good to use in gene therapy?
- small - can get into cells easily
- easy to administer
- not recognised by immune system so the immune response is not triggered
What is the gene therapy for SCID?
The T-lymphocytes are removed from the patients bone marrow and introduced to an ADA-modified virus. This is allowed to infect the T-lymphocytes and then the cells are put back into the patient.
No transfusions are needed.
What is the drawback to SCID gene therapy?
Before: It is not a permanent solution; transfusions are needed every 3-5 months.
Now due to new AAV virus: The virus does not insert its genes (inc. the corrected ADA gene) into the hosts genome, so it is not passed on to daughter cells when the cell divides.
What is the gene therapy for CF?
A virus which has the CFTR-modified gene is aspirated through an inhaler.
Why is the gene therpay for CF better than for SCID?
It is less invasive, and easier to administer because the target cells are an external cells of the lungs, which are easier to access. Bone marrow is not easy to access.
Is the gene therapy for CF permanent?
No.
Pros to gene therapy?
- helps people live normal life and have normal lifespan
Cons to gene therapy?
- not permanent
- expensive to research for limited successful results
- side effects (e.g. leukaemia in SCID treatment)
- tiny minority have benefitted
- changing genes is unethical; idea of humans who have these conditions need to be improved
- only for recessive conditions
Why is gene therapy not permanent
Specialised cells do not undergo mitosis, so the modified gene is not passed onto daughter cells
What does hybrdise mean?
in a microarray
process of binding of cDNA complementary bases to the ssDNA probes in microarray
