Topic 5 - Recombinant DNA technology 2 Flashcards
What is YAC? Structure…?
Yeast artificial chromosomes
Centromere, telomere (DNA sequence ends), autonomously replicating sequence (ARS) (similar to origin of replication in bacterial plasmids) - allows DNA sequences to be replicated.
What can the length of the telomere tell us about someone?
Good estimate of their age (get shorter in old age)
How many bp can YAK hold?
hold up to 200-1500k bp
Animal genes can hold how many bp?
can be >200k bp
What is BAC?
Bacterial Artificial Chromosome
Contain selection system eg. lacZ system
Carry up to 25% of bacterial chromosome
How many bp can YAK hold?
100-300k bp
The F-plasmid (fertility plasmid) & low copy number is associated with …?
BAC
Getting BAC into bacteria, what process is used?
electroporation (transformation) electrical shock very stable (DNA maintained normally in host)
Retrovirus based vectors are used for…?
for genetically engineering mammalian cells by incorporating DNA into the chromosome using a retrovirus
A bit more on retrovirus based vector…?
- two single-stranded RNA molecules joined at 5’ ends
- once in cell, uses reverse transcriptase to convert RNA -> DNA -> stably integrates into cell chromosome
What is a pro-virus?
Once the DNA has been incorporated into the chromosome = pro-virus
What is a lentiviral vector?
a special retroviral vector that can infect dividing or non-dividing cells
based on HIV1
Difference between lentiviral vectors & retroviral?
lentiviral vectors infect dividing or non-dividing cells, retroviral vectors only infect dividing cells
During engineering, hat are the 2 different types of retriviral vectors produced?
replication competent retriviral vectors
replication incompetent retriviral vectors (cannot replicate in cells.) why? delivers DNA to cell but does not produce more virus & infections of more cells - safer option for use in animals
Why are viruses said to be very species specific? ie. why would a human adenovirus not affect mouse cells or equine cells?
due to the structure of the proteins on the outside of the virus, organisms have special receptors for those specific proteins
Adenovirus properties…?
broad host range infect cells efficiently & easily pathogenicity low but frequent immunity effects in respiratory tract potential for gene therapy eg. cystic fibrosis
Learn about cystic fibrosis…
deficiency in ion transporter on surface of epithelial cells in lung -> ions out of cells -> pulls water thru -> ++mucous production
Caused by defective gene
Also causes problems with digestion
Why engineer eukaryotic cells…?
mammalian cells used to produce eukaryotic gene products, often posttranslational mods. (glycosylation, cleavage, phosphorylation) which E. coli cannot do
Which animal proteins can only be produced in animal cells…?
- factor VIII - blood clotting factor
- beta-interferon - anti-cancer protein
- growth hormone
- erythropoietin
Cell transformation method in bacteria…?
Treat cells with salts (CaCl, rubidium) followed by heat shock
Electroporation - short electrical pulse across cells in suspension + high voltage & low current -> makes transient pores in membrane -> DNA moves into cells
Cell transformation method in eukaryotes…?
Fungi & yeasts
Electroporation or chemicals like polyethyleneglycol (PEG) that interfere with membrane integrity -> allows DNA into cells
Cells can be cultured to regenerate cell walls and select for AB resistance genes same as bacteria
What is biolistics…?
used to fire DNA into organisms via:
gene gun - usually gold particles coated with DNA (4µm diameter). Fired at cells in vacuum -> nuclei
What is a liposome?
spherical membrane circles (lipid bilayers) formed in solution that incorporates DNA into lipid molecule to move DNA into cells (transfection) membrane fuses with cell -> DNA gets delivered into cell (lipofectamine)
Re. transformation, what does calcium phosphate do?
precipitates DNA onto surface of cell in culture
What is microinjection?
Process in transformation of multicellular animals, injecting embryo directly with DNA (into male pro-nucleus of fertilised egg)
What are reporter genes?
genes used to look at how genes are expressed in cells
expression easily detected (eg. lacZ & GFP genes)
luciferin
What is luciferase?
enzyme which uses compound produced in fireflies called luciferin -> with ATP to produce light. reporter gene producing light (eg. fireflies, glow worms, various fungi & bacteria)
Where does GFP come from?
jellyfish Aqueorea victoria
altered structure of GFP to give other colours
GFP fluoresces under which light? What other proteins can be used for genetic modification?
blue/UV light
RFP (red) or YFP (yellow)
Does GFP require substrate?
no - intrinsically fluorescent
What are the applications of using GFP & other reporter genes? Eg. transcriptional reporter..?
Way of looking at gene expression and how that’s controlled in a cell using GFP & reporter genes by fusing GFP with promoter for insulin gene…
Or if DNA encoding GFP fused with DNA encoding tubulin gene in a plasmid
Why would you use Southern blotting (DNA hybridisation)
Locate presence of particular gene in large pieces of DNA eg. insulin gene in larger clone
Or to locate similar genes in other organisms. ie. is insulin gene present in horses, kangaroos etc. is coding sequence different? is protein sequence different?
What does Southern blotting tell us?
What size a particular gene is & how many copies there are in a particular genome. eg. several copies of genes like insulin & where they are
What is hybridisation?
The ability of single stranded molecules to base pair with each other. eg. DNA from blood -> heat treat (90d) break DNA -> cool it down to 56-60d slowly -> strands start to reform finding their complimentary partner = hybridisation
Southern Blotting involves…?
Taking DNA from an agarose gel (seperated via electrophoresis) & blotting it onto a solid nylon or nitrocellulose membrane
Give example of when you would use Southern blotting
If you take blood, extract DNA, cut up with restriction enzymes from wallabies of same species in different parts of Aus. Looking for any genetic diversity between the different wallabies at different locations. Separate fragments via electrophoresis on basis of size -> look similar (not much difference), but looking at genetic variation around insulin gene using DNA isolated from humans. denature gel by sodium hydroxide (NaOH) -> DNA single stranded
What is the setup in a Southern blot…how does it work?
Tray with buffer (high salt), sponge, filter paper, gel, nitrocellulose paper, tissues or 2-3 nappies (absorbant material), weight. Fluid gets drawn from bottom to top into absorbant material. DNA moved thru gel into membrane (binds DNA under high salt conditions). Thus nylon membrane contain absorbed DNA on it. Then hybridisation…
Hybridisation…?
Immobilise DNA by UV light (or bake at 200d for 30min). Then cDNA labelled with radioactive 32P - probe. Probe denatured (heat or NaOH) & added to filter at 65d -> DNA strands find complimentary strand on filter & hybridise (stick) to them -> washed with reduced salt to remove excess probe. Xray film place over filter in xray cassette for 1-2 days -> emit radiation & expose xray film leaving pattern of bands where DNA called (autoradiograph).
If there was no genetic variation in species on Southern blot autoradiograph, what would we see on the autograph?
only a single band
If there was genetic variation in species on Southern blot autoradiograph, what would we see on the autograph?
might change position of restriction endoneclease (EcoR1 site) -> different bands eg. 8.7k bp vs 9.2k bp
First use of Southern blotting & hybridisation?
DNA fingerprinting (OJ Simpson case)
What does Northern blotting involve? What does it tell us?
separation of RNA molecules on agarose gel
Tells us how much RNA or transcript is present in a cell at any given time & size of that RNA (transcript) in different species
when & where genes are expressed
What is PCR?
Polymerase chain reaction
Way of obtaining very specific piece of DNA and amplifying it
What bacterium is used?
Thermophilus aquaticus
A bit more about PCR…
uses thermophilic polymerases (eg. TAQ)
Short synthetic pieces of single stranded DNA 10-30 bases long (primers)
requires very small amounts (nanograms) of DNA -> results in microgram quantities of target
Steps in PCR…?
- denaturation (92-94d) for 30 sec -1 min (DNA + TAQ + nucleotides + primers)
- primer annealing (cool to 25-65d) 60 sec -> primers stick to nucleotides (complimentary regions)
- Elongation/extension (up to 72d) TAQ polymerase begin to synthesise DNA (only on 3’ hydroxyl group from 5’ to 3’ direction) -> double the amount of DNA in 1 cycle -> repeat 40 times
What repeats the cycles? Amplification is …?
thermocycler
exponential
What is RT-PCR? What is it used for?
Reverse transcription PCR
used to look for the presence & level of mRNA within cells by converting RNA to cDNA & amplifying DNA
what is the limiting factor of producing DNA in RT-PCR? how do we avoid this? What method could we use instead?
run out of nucleotides in test tube or primers
reduce number of
What does Real Time PCR do?
quantitatively detects different amounts of DNA within a cell by the presence of DNA sequence & fluorescence as its being amplified (in each cycle 1-40)
Who is Fred Sanger?
guy who developed DNA sequencing method - dideoxy chain termination method
What is required for the dideoxy chain termination method?
- template (cloned fragment)
- oligo primer (20-30 bp)
need dNTP’s (dATP, dGTP, dCTP & dTTP) & corresponding didioxynucleotide triphosphates (ddNTP’s) at [low] - DNA polymerase
What colour is dATP, dGTP, dCTP & dTTP respectively?
green, yellow, blue, orange
What do ddNTP’s do in a reaction?
terminates the nucleotide chain once it gets incorporated into it because of it’s lack of a hydroxyl group on the 3’ carbon
A bit more about Sanger sequencing…?
Uses electrophoresis of DNA sample in thin poly acrylamide gel
can resolve differences in base pairs
sequence read from bottom
What is automated sequencing? (Sanger sequencing) ie. terminating 3’ base processing…
- dNTP labelled with fluorescent dye
- run all 4 reactions in 1 tube on 1 gel
- lazer detector determines order of colour thus sequence of fragment DNA
- 800 bp DNA can be determined per single reaction & lane of gel
3 steps in automated sequencing?
- generate nested array of fragments fluoresced & terminated at 3’ base
- separate by electroporesis
- scanning lazer detection -> into sequence data by computer (eg. orange light = G base)
First array used for gene expression?
macro array
Macroarray…?
high density filter
12cm x 8cm
2400 clones by membrane radioactive labelling
1 experimental condition
Microarray…?
glass slides 5.4cm x 0.9cm 10000 clones by slide fluorescent labelling 2 experimental condition
oligonucleotide chips…?
1.28cm x 1.28cm
300k oligonucleotides by slide
fluorescent labelling
1 experimental condition
Summary of arrays…
can analyse many genes in one experiment
known gene or mRNA sequence immobilised on substrate (filter, glass slide, silicon chip)
can label cDNA & DNA and hybridise to these
expression of many genes can be observed (eg. yeast genes, E. coli genes, human cDNA, bovine cDNA’s)
What genes are expressed in a certain liver tumour?
- isolate cDNA from liver & immobilise on glass slide (probes)
- isolate mRNA from tumour (test) tissue & normal tissue (control) & label cDNA eg. Cy3 (green) for tumour, Cy5 (red) for normal
- mix & hybridise to glass slide
- wash & scan to quantify amount of fluor. Then look at ratio of red to green to determine relative level of expression
