methods 6

Question 1

throughput of reporter genes

Answer 1

1-10

Question 2

throughput of northern blots

Answer 2

1-10

Question 3

throughput of in situ hybridization

Answer 3

1-10

Question 4

throughput of rtPCR/qPCR

Answer 4

1-100

Question 5

throughput of ddPCR

Answer 5

1-100

Question 6

throughput of nanostrings

Answer 6

10-800

Question 7

throughput of microarrays (cDNA and oligo)

Answer 7

10-1000s

Question 8

throughput of next gen seq (RNA-seq)

Answer 8

1000s

Question 9

How would you apply microarrays to gene discovery/ annotation? What DNA would need to be spotted (or synthesized) on the array?

Answer 9

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)

Question 10

What does the term “library” mean in molecular genetics (as in “cDNA library”, “RNA-seq library”, etc.)?

Answer 10

the entirety of DNA is fragmented, historically was always transformed into bacteria vector now we use PCR

Question 11

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?

Answer 11

transgenic line with a tagged protein (which you would have to ensure acts like wildtype) and use an antibody against the tag

Question 12

Yeast-2-hybrid (Y2H) is prone to artifacts, with many false positives and false negatives. Why do you think this may be the case?

Answer 12

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

Question 13

explain whole genome tiling arrays

Answer 13

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

Question 14

what is a pull-down assay?

Answer 14

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

Question 15

what is mass spectrometry

Answer 15

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

Question 16

explain co-ip

Answer 16

affinity purification technique to study protein of interest using a primary and secondary antibody and magnetic beads, can id protein complexes

Question 17

explain FRET

Answer 17

“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

Question 18

what is bimolecular fluorescence complementation (BiFC)

Answer 18

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

Question 19

what does yeast 1 hybrid measure

Answer 19

if a protein (transcription factor) binds to a DNA sequence (promoter)

Question 20

how does yeast 1 hybrid work

Answer 20

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)

Question 21

EMSA measures what interactions

Answer 21

protein-DNA interactions

Question 22

how does EMSA work

Answer 22

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

Question 23

DNase I footprinting what does it test

Answer 23

protein-DNA interacts

Question 24

How does DNase I footprinting work?

Answer 24

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

Question 25

was does ChIP stand for? and what does it measure?

Answer 25

chromatin immunoprecipitation, measures protein - chromatin interactions (either with naked DNA or histones)

Question 26

how does chromatin immunoprecipitation work?

Answer 26

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

Question 27

what is ChIP-Chip

Answer 27

a combination of chromatin IP and microarray tech to identify isolated DNA fragments

Question 28

how to identify transcription factor binding sites genome wide?

Answer 28

ChIP-Chip and ChIP-Seq

Question 29

what does DAP-seq stand for

Answer 29

DNA affinity purification

Question 30

how does DAP-seq work?

Answer 30

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

Question 31

what is “naked” DNA

Answer 31

DNA without histones

Question 32

What interactions does yeast-3 hybrid measure

Answer 32

protein-RNA

Question 33

explain the components of a yeast-3 hybrid

Answer 33

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

Question 34

name of the DNA binding domain of yeast 3 hybrid

Answer 34

LexA

Question 35

what is an aptamer and give an example of method that uses them

Answer 35

A folded ssRNA (or ssDNA) molecule that’s specifically recognized by a protein, yeast-3 hybrids

Question 36

Explain RNA EMSO

Answer 36

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