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Flashcards in AMB9 Deck (13)
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1
Q

What are the 6 check points used to moniter the control of gene expression

A
  1. transcriptional control
  2. rna processing control
    3 rna transport control
    4 translation control
    5 mrna degradation control
    6 protein activity control
2
Q

what is run of transcription

A

a method of measure the rate of transcription
conducted in vitro to identify transcription start site of a specific promoter along with its accuracy adn rate of transcriprtion.

3
Q

describe run off transcription

A

nuclei isolated - as not more ntps without cytoplasm = takes a snap shot of transcriptional activity in that nuclei

  1. dna with gene being actively transcribed will have a number of RNAPs on gene that are making copies
  2. radiolabelled UTPs (alpha 32 phosphate) are added
  3. UTP will be incorperated in transcripts when RNAPs start going again
  4. addition of nts allow rnaps to finish transcibing
  5. genes that are more transcriiptionally active = get more radiolablled transcripts.
4
Q

how are reporter genes used to measure transcriptional activity?

A

fused to promoter/enhancer and introduced into cells being studied, then promoter/enhancer determined by assaying reporter.
eg. luciferase gene can be uased as reporter and assayed sensitively and quantitatively an also be used to gain info of spatial distribution.

5
Q

what is the underlying assumption of using reporter genes to measure transcriptional acitivty?

A

that the reporter will not be subject to any regularition at any stages.

6
Q

what is in situ hybridisation?

A

technique similar to northern blotting except rna is not extracted from tissues, instead labelled probes are hybridised directly onto histological preps.
- eg, antisense labelled RNA probe, intensity of label indicate distribution and relative density of target rna.

7
Q

Describe an RNA protection assay

A

no longer used method
liquid phase hybridisation followed by RNAse digestion with enzymes that specifically target ssRNA. protected probes will be visible on gel and autoradiography
intensity of bands gives an idea of how abundant target rna is.

8
Q

describe qualitative pcr

A

sybergreen used to detect amplimers - dye binds to dsDNA and fluoresces when this happens
production of syber green pos material in pcr control sample with only water presetnt are there as products made that are not the result of amplification - post amplification analysis therefore required to avoid false interpretation.

9
Q

how can you reduce amplifcation of unwanted non target dna in pcr

A

qpcr using fret.

10
Q

describe qpcr using FRET

A
  1. 2 oligonts that target seq between amplification primer added with flourescent dyes bound to 3’ and 5’ end respectively. one red one green.
  2. blue light excites green dye
  3. when oligo binds to target and probe with red dye does too the close proximity causes energy absrobed by the green fluorophore to be transfered by fluorescent resonance energy transfer to neighbouring red dye so it will emit red fluorescence.
  4. amount of red fluorescent there is proportional to amount of target molecule
11
Q

what are microarrays used for?

A

to pinpoint differences in gene expression between different cell types

12
Q

what is a microarray?

A

a glass slide containing thousands of spots of DNA, each spot contains many strands of DNA that which corresponds to a single gene - can hybridise to cDNA or RNA
(essentially reverse northern blot)

13
Q

how does a microarray work?

A

probe:target hybridisation is usually detected and quantified by detection of a fluorophore can see which genes are turned on or not by if fluorophore flashes froor particular gene
probe is on solid surface. RNA is extracted from living tissue