BIOL 114 Flashcards
How do you obtain DNA without introns?
You take mRNA from lysed cells and use reverse transcriptase to make cDNA and Degrade the RNA with RNAse H so you can synthesize a whole DNA using primers, nucleotides and DNA polymerase.
How are genes cloned in bacteria?
A fragment of DNA is inserted into a circular DNA vector which is inserted into a bacterium (usually) which replicates the vector and the cells
How do you identify bacteria that have taken in a circular DNA vector?
The vector contains an antibiotic resistance gene and so any bacteria that survive said antibiotic will contain the DNA
What are restriction enzymes?
Enzymes that cleave DNA at specific sites in the nucleotide sequence
What does DNA ligase do?
It joins together DNA fragments by ligating the phosphodiester backbone
What are the conditions that make bacteria uptake DNA more efficiently?
Soaking in ice cold salt solution and then incubated at 42 degrees for 2 minutes
How do you minimise the chance of vector religation in bacteria?
Using phosphatase to remove 5’ phosphate groups
How do you identify cells that contain recombinant DNA after the antibiotic treatment?
Using a marker gene such as LacZ’ which makes an enzme (Beta-galactosidase) that converts X-gal into a blue coloured product.
You then put the gene of interest inbetween that gene so that if a blue product is produces then the target gene is not present to interupt so you can get rid of any blue colonies.
In electrophoersis how does the % agarose gel affect the resolution of DNA molecules that can be seperated?
Using higher percentages increases the resolution (smaller base pair sequences can be seperated)
If a healthy person prpduces 2 strips on electophporesis and a diseased person only produces one when cut with the same restriction enzyme what can you tell by this?
That a mutation has occured where the restriction enzyme would normally cut
How is strand synthesis stopped in sanger sequencing?
ddNTP is added which terminates the synthesis.
What sequencing technique was used to obtain the first human genome draft?
The chain-termination (sanger) method
What techniques are used for measuring mRNA?
Northern Blotting
RT-PCR
Microarray (genechip) techniques
Since all mRNA have a poly-A tail what does a poly-dT primer do when reverse transcriptase added?
It converts all the mRNA in the cell into cDNA unless a specific primer is added.
How is analysis of mRNA done to find gene expression (both current and previous methods)
Previous method: Microarray with flouresence labeled cDNA
Current: RNA sqeuencing which allows large groups of genes to be studied
How does nanopore sequencing work?
Nanopores embedded in synthetic membranes have ionic current measured as they pass through and when DNA passes through these nanopores it disrupts the current in specific ways based on the base pair passing through therefore this can be used to determine nucleotide sequence
Whats the difference between genomic libraries and cDNA libraries?
Genomic contains non-coding regions and is used to reconstitute genomes to study gene function
cDNA libraries only contains exons from mRNA and is used to reconstitute proteomes to study protein structure
What is hybridization?
Locating cDNA clone using a nucleic acid probe that consists of 100-1000 bases specific to the target cDNA that is labelled so it can be identified.
What types of probes are used in hybridization?
Homology probe: Corresponding DNA sequence from a related organism so DNA is almost identical)
Degenerate oligonucleotides: Degenerated DNA sequence from a known protein sequence. This can either be multiple labelled oligonucleotides for each option or using inosine-based artificial nucleotides where there is degeneracy in the code
What labels are used in hybridization?
Radioactive labels (phosphorus)
Non-radioactive labels (more complex to detect uch as DIG-specified antibody detection)
What is immunodetection?
Using antibodies to recognise protein produced by target cDNA
Proteins are transferred to nitrocellulose membrane (every protein binds to it) and add the antibodies to recognise them (after blocking any free nitrocellulose spots).
What is an expression system for recombinant proteins and what are some considerations to make when choosing one?
The host cell where you are making your recombinant proteins
Considerations:
Size
Solubility (membrane proteins wont be easily recovered from bacteria)
Post-translational modifications (bacteria can’t do this)
What can be optimised in E. coli expression?
The strain
The plasmid vector
The coding cDNA sequence inserted
How does optimising codon usage improve translation rate?
Degeneracy leads to codon bias where some codons are expressed more frequently so using these will improve rate and yield
How does incorporating N-terminal fusions help optimise recombinant proteins?
Improves translation rate by correcting codon bias
Increases stability and protects against degredation
Facilitates purification
What is micropropagation?
A method used to reproduce plants without seed
Done using an explant cultured under sterile conditions to promote regeneration of a whole plant using the plants totipotency.
What are the steps of regeneration by organogenesis?
Isolation of explant
Callus production on nutrient medium with auxin and cytokinin
Organogenesis stage 1 - shoot growth (cytokinin)
Organogenesis stage 2 - root growth (auxin)
What is somatic embryogenesis?
Development of embryos and plants directly from somatic cells (difficult in most species)
What are cell suspension cultures used for?
Reseach (conventient to obtain homogenous mass of cells)
Commercial (secondary metabolites and other compounds e.g antimicrobials, vitamins and good flavours)
What is somaclonal variation?
Phenotypic variability between individual plants from a plant tissue culture
What is somatic hybridization and what are protoplasts?
Protoplasts are plant cells without cell walls.
Somatic hybridization is the production of hybrid cells between sexually incompatible species.
What are the main steps in production of a trangenic (genetically edited) plant?
1) DNA construct production with gene of interest
2) Transformation of plant cells
3) Selection of transformed cell with marker
4) Regeneration of whole plants from transformed cells
What methods are used to deliver DNA into plant cells?
Naked DNA delivery - DNA coated particles (such as gold) shot into plant cells at high velocity to see which survive and if any DNA sticks or shocked with high voltage electricity to make plasma membrane permeable to DNA before resealing (usually used with protoplasts)
Natural Delivery - using plant pathogens with target DNA (T-DNA) added to transfer that DNA into the plant alongside the virus DNA
What are targets for genetic modification of plants (biotechnology)
Agronomic:
Yiels, Herbicide tolerance, Pest/disease resistance, Abiotic stress resistance, reproduction
Quality:
Processing, Shelf-life, Nutrition, Anti-nutritional reduction
Novel products:
Oils, Proteins, Polymers
Biofuels
What are the benefits and negatives of herbicide resistance?
Pros:
Enhanced crop production
Encourages “no-till” agriculture reduces soil erosion and lowers fuel costs
Cons:
Increased use of specific herbicides
Possible reduction of biodiversity
How does incesct resistance work (BT)
Modifys a plant to produce a Bt toxin produced by bacteria that is lethal to insects as after being broken down it binds to cell receptors and induces cell death.
What are the pros and cons of insect resistance?
Pros:
Cheaper alternative than pesticide
Reduced environmental impact
Improved farmer health
Improved food quality
Cons:
Selection pressure for insects
Possibly effects non-target insects (e.g pollinators)
What are the pros and cons of increased nutritional quality?
Pros:
Potential to widely battle malnutrition and has saved millions from death or blindness (golden rice)
Cons:
Lowers diversity in agriculture and reinforces dependence on rice (in the case of golden rice)
How is gene editing done?
Either though whole gene insertions with markers or through CRISPR-Cas9 which allows targeted editing at specific sequences.
How are cells grown in culture?
Rich growth media,
Some from of rerum with growth factors (fetal bovine serum),
Sterile conditions,
Buffered pH and osmolarity,
Need solid growth surface
What are the stages of tissue culture?
Stage 1) primary culture (cells extracted from animal tissues and begin to grow in culture)
Stage 2) Cells grown in primary cell line in dish until space is filled with monolayer (observed with microscope) Then trypsin is added and cells are moved to fresh culture dishes.
Stage 3) ‘senesence’ Most cells die after about 50 replications.
Stage 4
How are indued pluripotent (iPS) stem cells made?
“stem cell” master regulater genes are introduced to precursor cells using retroviral cloning vector that reverts the cell into an iPS cell
What are the pros and cons for Inducted pluripotent stem cells?
Pros:
Fully differentiated cells
Fewer ethtical shenanigans
Can use patients own cells
Cons:
Technically challenging
Risk of cancer (chance of shoving a super mega tumor into patient)
What is an alternative to Induced pluripotent stem cells without using embryos?
Direct reprogramming a cell from one cell type to another.
What is a trangenic animal?
An animal that is carrying an additional gene it doesn’t normally possess.
How are transgenic animals created?
Foreign DNA is injected into the pronuclei of a fertilised egg (usually the larger one)
How are transgenic animals identified?
PCR is used on the offspring to analyse for transgene presence
What is a gene knockout?
A wild type gene is replaced with an inactive or mutant gene.
It is controlled exactly where the change is made.
Very low success rate.
How does gene knockout work?
Homologous recombination:
Target gene is replaced by neomycin resistant replacement gene (TK gene is not in new gene)
-Resistant to G-418 and ganiciclovir
Nonhomologous recombination:
Gene is randomly inserted in a region outside of target and TK gene is also inserted so it is no longer resistant to ganciclovir.
Why aren’t adenoviruses used for gene therapy and what is the alternative?
They elicit an immune response that can be severe and possibly fatal for patients.
Inserts randomly into genome and may inversely affect patient
Adeno associated viruses are used insstead as they don’t elicit much immune response.
Vectors developed to control protein expression
What diseases are targets for gene therapy?
Haemophilia
Cystic fibrosis
Duchenne musular dystrophy
Sickle cell anaemia
Cancer
Rheumatoid arthritis
Erythropoietin for kidney patients
What DNA tests for diseases are there?
Neonatal screening - early detection
Carrier scrning - shows the risk using the genes of the parents
Predictive screening - Identification of late onset disorders
pre-implaantation sreening - high risk embryo screenig
Invasive foetal screening - if high risk
Non-invasive (uses mothers blood sample)
How can repeat expansion diseases be diagnosed with PCR
Extract DNA flank repeating sequence and seperated with electrophoresis
How to identify a change in base at a particular DNA location?
Use a primer (if it is expected base then primer will fit otherwise it wont extend as it is mismatched)
What is a single nucleotide polymorphism?
Its a polymorphism in a single nucleotide that corrolates to a disease (not neccessarily causing it) and can be identified easily to determine if someone is at risk of the disease.
How are relavent single nucleotide polymorphisms found?
Using a Genome-wide assocition study (GWAS)
Genomes of many people with genetic condiions tested for millions of SNPs and highest frequency of statistically sigificant SNPs compared to unaffected people is used
What technique is used to identify chromosmal diseases?
FISH (Fluorescent in situ hybridisation)
What are the problems with current genetic tests?
Restriced to limited number if diease alleles and prior knowledge of these alleles is needed for testing.
What are causes of infertility in males and females?
Males:
Sperm quality
Duct blockage
Lack of gonadal tissue
STD/illness
Sperm destruction
Low testosterone
Medications
Females:
Age (of oocyte)
Problems with oocyte maturation and/or ovulation
Lack of gonadal tissue
Lack of uterus or different structure
Uterine development stage
uterine tube blockage
Stress
Scarring (uterine)
Endometriosis
Cervical mucus
Medication
BMI
Why is secondary infertility more common?
Age
Underlying health
Weight
Scarring from previous pregnancies
Fibroids
Damage from infections
How are eggs retrieved for IVF?
Hormonal treatment supresses natural cycle
Gn RH agonists inhibit pituitary function
GnRH antagonists inhibit LH/FSH
Exogenous hormones stimulate ovulation
Monitoring of oocytes via ultrasound
Oocytes harvested transvaginally
How is a good quality egg identified?
Large egg with polar body
Day one: 2 pronuclei
Day two: 4 cells
Day 3: 6-8 cells
Day 5 : Blastocyst with good trophoblast and inncer cell mass
Day 6: Blastocyst hatches
What are problems with IVF?
Expensive and invasive
Increased risk of ectopic pregnancy
Risk of ovarian hyperstimulation syndrome
What are the methods of sperm extraction?
PESA
MESA
TESA
TESE
Micro-TESE
What are the limitations of multi-locus fingerprinting method?
The bands produced can’t be assigned to any particular locus
DNA fingerprint not readil amenable to stats analysis (hampers use in courts)
How does multiplex PCR work?
Extracted DNA is amplified containing STRs with 10 pairs of rimers with flourescent dyes to allow identification when seperatated in high resolution polyacrylamide gel.
What can mtDNA be used for?
Useful in old or degraded samples
can be used to determine ethnic origin, some physical characteristics
Surname (if male via Y chromosome markers)
Face prediction
