methods 6 Flashcards
throughput of reporter genes
1-10
throughput of northern blots
1-10
throughput of in situ hybridization
1-10
throughput of rtPCR/qPCR
1-100
throughput of ddPCR
1-100
throughput of nanostrings
10-800
throughput of microarrays (cDNA and oligo)
10-1000s
throughput of next gen seq (RNA-seq)
1000s
How would you apply microarrays to gene discovery/ annotation? What DNA would need to be spotted (or synthesized) on the array?
tiling arrays can be used to help id fragments of DNA that are expressed vs not expressed (by hybridizing with RNA), also can find chromosomal alterations (by hybridizing with DNA)
What does the term “library” mean in molecular genetics (as in “cDNA library”, “RNA-seq library”, etc.)?
the entirety of DNA is fragmented, historically was always transformed into bacteria vector now we use PCR
If you want to compare the abundance of a protein of interest between two tissues, but do not have an antibody against that protein, what are your options?
transgenic line with a tagged protein (which you would have to ensure acts like wildtype) and use an antibody against the tag
Yeast-2-hybrid (Y2H) is prone to artifacts, with many false positives and false negatives. Why do you think this may be the case?
in yeast the “context” is different from normal cells, proteins usually separate in native cells like mito vs nuc may be together in yeast
protein may need to be in a complex to function
protein may need post translational modifications
explain whole genome tiling arrays
RNAs are isolated, reverse transcribed, labeled and hybridized to a tiling array => the spots/tiles that light up indicate the parts of the genome being transcribed => experimental gene annotation
what is a pull-down assay?
in vitro technique to study protein protein interactions
bait is bound to a resin used to purify protein of interest, resin is washed away then bait is eluted, prey is identified with mass spectrometry
what is mass spectrometry
MS is an analytical technique that can be used to infer the identity of the protein(s) in a sample by measuring the mass-to-charge ratio of ionized protein fragments
explain co-ip
affinity purification technique to study protein of interest using a primary and secondary antibody and magnetic beads, can id protein complexes
explain FRET
“ratiometric”
2 fluorescent proteins which transfer energy between one another if close together, the donor molecule is excited by a laser and the donor molecule is transfered to an acceptor molecule + an emission occurs from the acceptor molecule
fuse a gene of interest to one fluorescent protein (CFP) another gene of interest to another fluorescent protein (YFP) and test their interaction
what is bimolecular fluorescence complementation (BiFC)
a fluorescent protein is split into 2 between 2 proteins, so if the two proteins interact they’ll fluoresce.
gives more of a “yes or no” answer
easier method than fret if you don’t have the best scope
what does yeast 1 hybrid measure
if a protein (transcription factor) binds to a DNA sequence (promoter)
how does yeast 1 hybrid work
a transcription factor of interest is tested in yeast for the ability to bind to the promoter region, the TF is fused to an activation domain, the bait (sequence you’re testing if TF binds to) is subcloned upstream of a reporter gene (auxotrophic or colorimetric marker)
EMSA measures what interactions
protein-DNA interactions
how does EMSA work
a radio or fluorescently labeled DNA fragment is incubated with purified protein or lysate, run on a gel and see if it runs at different weights
DNase I footprinting what does it test
protein-DNA interacts
How does DNase I footprinting work?
label the end of the DNA, incubate with protein of interest, DNase digest (it won’t cut where protein is bound, leaves a footprint), run on a polyacrylamide gel and a “footprint” will be left with missing bands
was does ChIP stand for? and what does it measure?
chromatin immunoprecipitation, measures protein - chromatin interactions (either with naked DNA or histones)
how does chromatin immunoprecipitation work?
crosslink cells with UV or drug
shear chromatin
pull down fragments with antibodies against protein of interest
purify DNA that was attached to protein and follow up by PCR, sequencing, or microarrays
what is ChIP-Chip
a combination of chromatin IP and microarray tech to identify isolated DNA fragments
how to identify transcription factor binding sites genome wide?
ChIP-Chip and ChIP-Seq
what does DAP-seq stand for
DNA affinity purification
how does DAP-seq work?
Similar idea to ChIP-seq.
genomic DNA is fragmented and adaptors are ligated to end, co-incubated with a tagged protein of interest, the protein/DNA complex is affinity purified, and the DNA is eluted, PCR amplified using adaptor, and sequenced
what is “naked” DNA
DNA without histones
What interactions does yeast-3 hybrid measure
protein-RNA
explain the components of a yeast-3 hybrid
2 hybrid proteins:
hybrid protein 1 has a DNA binding (LexA)/RNA binding domain
hybrid protein 2 has an RNA binding/activation domain.
RNA contains an aptamer, a region which allows binding to RNA binding domain of hybrid protein 1
DNA binding site is upstream of reporter gene
Hybrid RNA is bound to hybrid protein 1, if hybrid protein 2 interacts with the RN the activation domain will be near enough the reporter gene to activate transcription
name of the DNA binding domain of yeast 3 hybrid
LexA
what is an aptamer and give an example of method that uses them
A folded ssRNA (or ssDNA) molecule that’s specifically recognized by a protein, yeast-3 hybrids
Explain RNA EMSO
RNA is isolated, labeled, co-incubated with a protein of interest, and complex is run on a gel. If band doesn’t run as far you know RNA is bound by protein
