Molecular Biotech Quiz 2
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- What are the different biotech applications of microorganisms?
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1. production of whole cells (biomass)
2. production of low-molecular-weight compounds such as primary metabloltes, secondary metabolites, and transformationof organic compounds by non-growing cells
3. Production of high-molecular-weight compounds such as polysacharides, lipids, and proteins
4. Processed dependant on general microbial metabolism such as the degredation/oxydation og effluent and noxious wastes or mineral extraction. - What are examples of low-molecular-weight compounds that can be produced by microorganisms
- primary metabolites, secondary metabolites, transformation of organic comnpounds by non-growing cells
- what are examples of high molecular weight compounds that ban be produced by microorganisms?
- polysacharides, lipids, proteins
- What are examples of processes dependant on microbial metabolism?
- The degredation/oxydation of eefluents and noxious wates, mineralk extraction
- What are some major products of biological processing?
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bulk organics (non beverage ethanolacetone)
biomass (yeast)
organic acids (citric acid, lactic acid)
amino acids
microbial transformations (steroids)
antibiotics
extracellular(xanathan gum) polysacharides - Explain primary and secondary metabolit production in relation to cell mass
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the production of primary metabolites closely follows the cell mass curve: it takes a certain number of cells to make a certain number of primary metabolite.
Secondary metabolites follow a different curve, one a certain cell mass is achieved, the cell makes an exponental amound of 2nd metabolite - What is the less costly to produce a 1st or 2nd metabolite? Why?
- 2nd because you dont need to keep growing cells to get porduct, one you achieve a certain cell mass the 2nd metabolite will be max produced.
- What are primary metabolites?
- They are needed for cell survival and energy.
- What factors determine purity?
- The more often a product is used, the larger the dosage, the pures it has to be.
- What three factors come into play when making product decisions about biotechnology?
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Are you the first to market?
/ \
/ \
low cost high quality(yeild of (purity)
product) - What is more economical, large scale or small scale production?
- large scale
- What are factors that affect the expression of clones genes?
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-promotor stength (including regulators)
-transcriptional termination
-secondary structure of mRNA
-translational initiation sequence (ribosomal binidng site)
-plasmid/gene copy number
-plasimd/gene stability
-fusion/signal sequences
-host cell physiology (choice, media, conditions, reactor etc) - What is a promotor?
- Where RNa polymerase binds to the gene
- What are 3 important signals for gene expression?
- Promotor, Ribosomal binding site, terminator
- What is the ribosomal binding site?
- point at which ribosome binds w mRNA
- When talking about promotors what are two important regions? What are they in reference to?
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In ecoli the -35 box and the -10 box
In animals various signals and the -25 box
These numbers are in reference to the site if transcription - When using an expression vector where is the gene of interested located?
- 3' (downstream) of Promotor and Ribosomal binding site
- What is important to remember about the promotor?
- It must be appropriate to the HOST system
- What does it mean when a protor is storng? weak?
- A strong promotor produces a lot of trancripts. A weak one produces few transcripts
- What does it mean when a gene is inducible? example?
- It is normaly off and can be turned on. The LAC operon contros an inducible gene?
- What does it mean when a gene is repressible? Example?
- It is normally on and can be repressed. The Trp promotor controls a repressible gene.
- What is characteristic of bacterial mRNA?
- It is polysistronic, one promotor controls the transcription of more than one gene.
- What is an operation?
- a region downstream of the promotor that controls the gene (for inducers, repressors)
- What is an operon?
- promotor, operator, structural genes, terminal
- What is a terminal
- Region where transcription ends
- what is an effector molecule?
- a molecule that attatches to a repressor so it cannot bind to DNA
- What is a repressor?
- A molecule that physically binds to DNA and physically blocks the transcription of DNA
- What is a corepressor?
- A molecule that binds to a repressor and allows it to bind to DNA, stopping transcription.
- When it transcription of the LAC opron highest?
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glucose concentration is LOW
lactose concentation is HIGH
cAMP (bound with CAP)concentration is HIGH - What determines the concentration of cAMP?
- WHen the cell in in a low energy state cAMP is high
- What are inducers of Lac promotor?
- lactose or IPTG
- What is IPTG
- looks like lactose but isnt metabloized
- What is CAP?
- Catabolite activator protein. It enhances transcription by enhancing the affinity of cAMP for promotor
- What can induce lac promotor?
- lactose or IPTG
- What it IPTG
- molecule that is recognized by cell as lactose but cannot be metabolized
- How does the trp promotor work?
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repressoble therefore always on ---> produces tryptophan
tryptophan negativly feedsback and binds to regulatory protein (inactive repressor) activating it and allowing it to bind to DNA --> transcription stops - WHat are some commonly used promotors. What changes their strength?
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lacUV5- varient of lac
Tac- repressed by lac repressor, reversed by IPTG or lactose
TRp- negatively regulated --> off with trp, on w/o trp or in presence of 3-indolearcrclic acid
PL- cI repressor protei of lamda phage; 28-30 degrees off, 42 degrees celc on - How does the PL promotor get regulated?
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pCI---> transcription and translation of cI gene
at 30 degrees active cI protein acts as repressor of the L operator--> no transcription
at 42 degrees inactivation of cI protein ---> no binding --> transcription of gene - Which promotor works best for what host?
- differnt promotors woek differently with diff hosts.
- What changes promotor strength?
- The region between -10 and -35
- What can be done to prevent promotor leakyness? example?
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a dual plasmid system:
cI gene under control of trp promotor in one plasmid. Target gene under control of pL promotor.
No trp in medium, cI protein is synthesized, binds to pL and blocks transcription of taget gene
add trp to medium---> trp promotor is repressed, no cI protein, Pl promotor is active, target gene transcribed - What is the signal that intiated translation?
- The ribosomal binding site.
- What are some characteristics of the RBS?
- 6-8 nts (UAAGGAGG) in mRNA that binds by molecular recognition to the small ribosomal subunit. It must be a propper distance away from the star codon (AUG)
- What should be avoided in mRNA? why?
- RNA secondary structures such as hairpins will cause a ribosome to fall of reducing protein expression
- WHAt are some strategies for increasing protein production/stability?
- tandem repeats, N-terminal modification of RNA, protease-deficient host strains, DNA integreation into host chrmsm
- What are tandem repreats?
- Multible copies of a gene cloned into a vector
- What adds to the srability of a protein? Why?
- N terminal modification protected against degredation by proteases. Added to gene at pcr or in vector.
- Why would you insert a gene into the chrmsm?
- improves stability of gene (plasmids can be lost)
- How is a gene inseted into a chrmsm?
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plasmid has regions of homoogy with known part of chrms that is not essencial to cell. Crossover events btw plasmid and chrmsm replace non essencial region with target gene.
Can use a dbl xover systme (homologous regions flank gene of interest) or single xover (whole plasmid is integrated) - What is a more succesful strategy: mulicopy plasimids, or gene integration into chrmsm?
- multiple copies of gene integrated into crhmsm give am activity.
- How can one check if a gene has been integrated into the chrmsm?
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Use marker:
Insert marker into chrmsm -- select for marker
then insert gene into marker region --> select for inactive marker - What is a signal sequence?
- a sequence at the N terminus of a polypeptide (or 5' end of gene) used for protein trafficking
- What is an N-terminus fusion
- something added to the 5' of gene (or N terminus) that will aid in purification
- Where are signal sequences placed? why?
- at 5' end b/c proteins are packaged while translated
- Where can a purification fusion be placed?
- either at 5' or 3' but 3' is better because you know that purified protiens are fully transcribed and translated.
- What can cleave a polypeptide?
- a protease the cleaves at sepecific amino acid sequemces
- What should you be able to do to signal sequences and fusions? why?
- cleave them off b/c final product must sometime be JUSt protein (FDA rules)
- What must one be aware of when insering a fusion protein? How do you overcome this?
- Reading frame. vectors with different # of nts to fix reding frame (one will work)
- Where is a fusion proten inserted?
- before Termination site
- What are some common fusion proteins?
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his tail (binds to nickle on colums)
trep tag (binds to streptavin)
GST [glutathion-S-transferate] binds to glutathione beads - What does cyanogen bromide do? How is it used?
- cleaves methionine residues. Methionine tag may be used to recover protein problem is: the protein cannot have other met or else will get cleaved!
- What are some methods for recovery of fusion proteins
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immunoaffinity (anitbodies)
chemicla method (met)
afinity chromatography (beads) - what are the benefits/limitation of immunoaffinity for purification?
- very accurate but also very expensive
- Other than purification how else can fusions be used?
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fusion with outer membrane surfance protein can be a screening tool (libraries) or display system
can either be at N terminus or at surface loops - What are some agents that can remove fusion proteins? Which are best? why?
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cyanogen bromide, formic acid, protesases
smaller chemicals are better because they are less expensive. enzymes work well but are more expensive - What is a characteristic of signal sequences?
- they are usually hydrophobic in the middle
- What are some differences between eukaryotic and prokaryotic expression vectors?
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Prokaryotes may include contaminants that cause inflamation or immune response in ppl
the product can be unstable bc of a lak of glycosylation
the protein may lack biological activity b/c of a lack of post translational mods
Eukaryotic system is more expensive and purification is more complex - What are some types of post trans mods?
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disulfide bons
glycosylation
amino acid mods (phosphorylation, sulphation) - What are some characteristis of euk expression vectors?
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usually shuttle vecotrs (most genetic manipulation in ecoli)
if not chromosomal than integrated into host chrmsm
must have euk select marker as well as euk ori and ecoli ori and markers - What are some features of yeast expression vector (saccharomyces cerevisiae)
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single cell
well known
easy to grow in sm or larg scale
STRONG PROMOTORS
NATURAL PLASMID
post trans mods (close enough)
secretes few proteins
GRAS by FDA - What are the 3 types of expression vectors for yeast?
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episomnal (plasmid)[YEps]
integrating [YIps}
YACs - What makes pichia pastoris sometimes a better choice than sacharomices as a euk express vector?
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high cell density (no toxicity bc no ethanol production)
even fewer secreted proteins
no hyperglycosilation
highly regulated gene (alcholol oxidase by methanol)
servisea has plasmid intability during up scale - other than yeast what are some other common euk express vecors?
- insect cell (baculovirus), chinese hampster ovary cells (mammalian )
- what are benefits of insect cell expression?
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strong polyhedrin promotor for viral protein coat
similar post trans mods
use of baculovirus to infect dells - How does an insect cell expression vector work?
- Transfer vecotor intgrated into chrms of virus( crossover at polyhedrin gene loss of gene
- How does one improve # of recoms in AcMNPV baculovirus?
- linearize before transfectipon.
- What is a bacmid?
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baculovisus- plasmid hybrid
Two plasids + viral genome
one plasmid with gene, polyhedrin region, markers etc another with tranpositional genes that facilitate donor plasmid integration into bacmid - What are some characteristics of mammalian expression vectors?
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extracromsmal
same post mods
CHO cells for long term stability
ORI from animal virus
promotors from human viruses or mammalian genes - What are some examples of Mammalian cell selectable markers?
- DHFR- methotrexate (MTX cells cannot grow inless make the enzyme = tranfected with vector
- What is IRES? when it it used? why?
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IRES= internal ribosomal entry site.
It is found in mam virus genomes and allows silultanious translation of diff proteins form polycis mRNA
Used to ensure same # of two polypep (for assembles protein) - What a 3 potential probs with expressin og foreign genes in ecoli?
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cannot exclase introns
premature termination
codon bias
RNA secons struct
reading frame - Pichia is often used as an expression vector in lieu of Saccharomyces because?
- no ethanol toxicity B. higher cell densities C. lower hyperglycosylation
- Feedback inhibition of metabolic pathways such as amino acid biosynthesis is beneficial to organisms because
- A. reduces ATP use B. promotes efficient use of enzymes C. improves survival
- The pL promoter is shut off (repressed) by:
- cI repressor protein
- Parts of a fermentation/downstream process include
- B. optimal growth conditions for engineered organism C. medium formulation D. product extraction
- The important control points in gene transcription and protein expression include:
- . terminator B. ribosomal binding site C. promoter
- Ribosomal binding sites optimized for protein production would:
- B. usually be <10 nucleotides in length C. be the location of translational start
- Signal sequences can help in protein production by:
- B directing proteins to subcellular organelles C. secretion of proteins outside of cell
- A western blot includes:
- anibody and protein
- Reading frame refers to:
- translation
- n agarose gel electrophoresis, the extent of DNA migration is dependent upon:
- B. size of DNA C. gel concentration D. voltage
- Examples of fusion systems include:
- A. his tail B. glutathione S-transferase C. streptavidin affinity peptide D. maltose binding protein
- Mammalian expression vectors contain
- A. mammalian selectable marker B. ori from animal viruses
- Stability of cloned genes can be improved by:
- A. incorporation into chromosome B. using antibiotic selective pressure
- Expression of cloned genes can be improved by:
- A. optimizing promoter strength B. avoiding mRNA secondary structure C. optimizing ribosomal binding site D. optimizing transcriptional termination site
- se of microorganisms and enzymes in pharmaceutical synthesis is due to:
- B. selective chemistry C. potential for genetic control of pathways
- Primary metabolites are different than secondary metabolites since they:
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A. are produced before the secondary B. they include glycolysis intermediates
D. they are metabolized more readily - Differences between a cloning vector and an expression vector include:
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A. ribosomal binding site
C. promoter D. terminator - For a regulatable promoter where an inducible gene is involved, in the absence of a regulatory chemical:
- transcript is made
- Anchorage dependent cells can be grown in:
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A. T-flasks
C. Roller bottles D. Hollow fiber systems - Scale up challenges for mammalian cells include:
- A. serum B. contamination from bacteria C. antibiotics D. oxygen transport
- What is fermentation?
- any process for the production of product by the mass culture or microorgainsms
- What are the 6 parts of a fermentation process?
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1. formulation of medium
(work about stock batches materials)
2. sterilization of medium and equiptment
3 Active pure cultures for inoculation of production vessel (every new run needs to start w same stock)
4. growth of organism under optimum conditions for product formation
5 extraction of product (purificiation)
6. disposal of effluents from process - Why must easch new run of fermanetation start with stock cells?
- b/c end product has no proof of gene stability and lack of mutation
- What are some fermentation issues?
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sterility
media (defined or nutrient rich)
oxygen pH
temp
batch, fed batch, contin
time
intracell or secreted
post trans mods - what is the diff btw defines and nutrient rich media?
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defines know exactly what is in it (for bateria and simple media)
nutrient rish dont know complonenents for euk - What determines batch/ fed batch. cont choice?
- purity and amnt of pruducr
- why is time important to fementation?
- more time = more product also more cell death
- What is diff btw imortal v primary cells
- immortal never stop dividing alwys use importal host cells
- What is anchorage dependant growth?
- cells only grow/divide if stuck to surface (usually euk cells)
- What is a spinner bottle used for?
- suspension culture
- what are different modes of fermentation?
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batch culture (one medium, cells grow, harvest)
continuous culture (fresh medium in on one end, large volume culture, contin growth, medium and some cells out at other end) - What are the scale up anchorage dependant cell methods
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roller bottle
microcarriers (small beads)
hollow-fibre apparatus - What are probs with scale up mam cultures?
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cost
quality serum (high cost)
contamin from bacteria bc of long cell division time of euk
anibiotics are costly and need to be purifies
special equipt for anchor dependant
more complex nutrition requirements
smaller pH window
isotonic w cell cytoplams
oxygen content - Why is fermentation scaled up stepwise?
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moderation of control easier
if direct put cells in large fermentor die b4 reach biomass
control changes at each step bc of changing demans of culture for oxygen diffusion changes - What are some general scale up probs?
- physical and chem apapmeters that influence cell chane with scale
- things that determine scale up expression:
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rule of thump: optimal conditions change with 10x increase of reactor volume
variables (teno,pH, rate of myxing, O2) - What is the diff btw batch, fed batch, cont?
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batch- no fresh growth medium (s curve cell growth)
fed batch- media added periodically, none removed (MOre sustained s curve)
cont- fresh media added, old removed (stable cell growth)
Yeild increases w fed batch from batch - What are the diff parts of a growth curve? where is protein production?
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1.lag
2. acceleration
3.*log dN/dt =uN
4.deceleration
5. stationary
6. death
protein production best at top of log growth - What is advantage of batch growth?
- predictablke growth at log growth can plan for extraction etc.
- What effects does plasmid copy have on metabolic load?
- more plasmids increases load and decrease growth rate
- What are the benefits/problems of fed batch over batch?
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benefits: extebds log and stationary phases
increases biomass and second metabolites
yeild increase
probs:
protease activity
monioting requirements - What are the benefits of cont vs batch?
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lower cost
smaller biorectors
smaller purification (downstream) equiptemet
avoids time btw rums
more uniform cells
probs:
up to 1000 hrs loss of plasmids (aviond with chrms integration)
sterility
composition of media potemtially variable - What does oxygen do>
- key role as reminal electron acceptor
- What is metabolic load?
- amount of energy it takes to make product
- Who funded HGP?
- dep of enegry
- when did the HGP start?
- 1988
- What are the difficulties of the HGP?
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high density of DNA
sheer size
simple repetative nauture of code
cost
infor manegement - Decribe Sanger sequencing
-
1977
PCR with dideoxys
originally checked with radioactive lables now with florescence in cappilarry gel and machine that can readout - what is average size of sanger eadout?
- 700bp
- What are the two approaches to genome sequencing?
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HGP - Bac based hierarchal shotgum
Celera- whole genome shotgun approach - what is difference between whole genome shotgun and hierachal shotgun approach?
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hieracrchal- make bac library
multiple ppl can work at smae time
Lower frequency of misassemblies, more practical for diploids, manages cloning bias, suited for international project
whole genome shotgun
whole genome broken apart
all reads assembled, computationally intensive - what are some informatics challenges whith genomics?
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Storage, back-up and curation of massive
amounts of DNA sequence data
⬢ Computationally intensive assembly process
⬢ Tracking of large numbers of biological samples
⬢ Coordination of sequencing effort across
multiple labs and countries
⬢ Data compatibility issues
⬢ Normalization of data quality - When construction a BAc lib how is a remplate constructed?
-
Solid Phase Reversible
Immobilization used for much of
the HGP & still in use at many
facilities
⬢ DNA can be reversibly bound to
the surface of functionalized
magnetic beads under the
appropriate buffer conditions
⬢ The beads are separated from
other cellular components and
washed
⬢ The DNA is released from the
beads by changing the buffer
solution - What is templi phi? how does it work?
-
tempiphi is a DNA amplification method (a DNA poly)
it is Highly processive single-subunit enzyme with stranddisplacement
activity
⬢ Proofreading functionality ensures amplification fidelity - When was HGP concluded sort of?
- 2003
- When dealing with infectious disease viruses how do you work with generic material?
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DONT use the infectious RNA
reverse tanscribe to DNA instead - What are the 4 new sequencing platforms?
- 454, solexa, helicos, polinies
- How does 454 work?
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– Amplified single molecules on beads
– Pyrosequencing (luciferase)
- when base binds with bead it light up
can work at low G+C content
input DNA doesn't have to be cloned
not good for homopolymersinfo for population genetics and diversity - How does soexa work?
-
immobilization on glass slides
termination reaction but terminators are removable - How does helicaose work
- no pcr less error
- What is illumina, how does it work?
-
⬢ Massively parallel SNP detection
⬢ Utilizes coated beads immobilized in fiber-optic
microarray
⬢ Each bead carries one oligo
species
⬢ Beads auto-assemble into
wells of the array
⬢ Beads carrying ~2000 different
oligo species can be loaded
onto a single array with ~ 25-
fold redundancy - What is the internation hap map project?
-
⬢ The goal of the International HapMap Project is
to describe the common patterns of human
DNA sequence variation by developing a
haplotype map of the human genome
⬢ The map enables researchers to identify genes
affecting health and disease and may help
explain the variability in responses to drugs and
environmental factors between individuals - what do microarays do
-
for gene expression studies
⬢ Microarrays enable the user to visualize the
expression levels of thousands of genes in
parallel
⬢ Affymetrix holds the patents on high-density
microarrays and are a leading company in the
field
⬢ Affy recently launched an array of all known
human genes on a single chip - What is the basis for function of a DNA microarray system – what is it, what does it tell you, and in general terms how does it work?
- Arrays of oligs – bind target and fluoresces – determine sequence or determine mRNA profiles
- ) How does 2D gel electrophoresis differ from more traditional gel electrophoresis and why is it used?
- First pH, then MW – separates proteins better – e.g. for proteomics
- a) What is a SNP and (b) why are they important in genomes?
- Single nt polymorphism, most common type of variation in human genomes, thus responsible for many phenotypic differences. Also useful in haplotype mapping for high resolution genotyping of individuals without having to sequence entire genome