L7: Gene Expression and Gene Expression Profiling

Question 1

Central Dogma

Answer 1

DNA => RNA => Protein

Question 2

How do you turn genes on and off

Answer 2

controlling transcription

Question 3

Define promoter and operator

Answer 3

promoter: DNA segment allowing gene to be transcribed
operator: part of DNA that turns gene on and off

Question 4

What does an operon include

Answer 4

operator
promoter
one or more structural gene

Question 5

Where are operons most common

Answer 5

prokaryotes

Question 6

What does a lac operon contain

Answer 6

three genes encoding for enzyme that break down lactose

Question 7

on and off of lac operon

Answer 7

off : no lactose, has repressor
on: lactose present

Question 8

What is transcription controlled by

Answer 8

regulatory DNA sequences and protein transcription factors
TATA box
enchancer + silencer
regulatory sequences

Question 9

What else is important in gene regulation

Answer 9

RNA processing

Question 10

What is a transcriptome

Answer 10

set of transcropts and relative expression levels

Question 11

what is the objective of gene expression profiling

Answer 11

moniter mRNA abundance and changes to understand gene function

Question 12

List 6 methods of gene profiling

Answer 12

1. northern blotting
2. RT PCR
3. cDNA
4. Microarray
5. Bulk RNA seq
6. Single-cell RNA seq

Question 13

What are two mechanisms of microarrays

Answer 13

hybridisation by cDNA spotted array or oligonucleotide array

Question 14

What is Bulk RNA seq

Answer 14

sequencing based on gene expression profiling

Question 15

Taq polyermase only works with DNA so how do we use it

Answer 15

reverse transcritpn RNA => cDNA

Question 16

How does SYBR green work, why use it, how

Answer 16

binds to ds DNA and emits like when illuminated at a wavelength
why: dsDNA intercalating dye, unspecific = optimal = cheap

how: apply => extend =>apply excitation

Question 17

What is taqMan probe chemistry

Answer 17

sequence specific
doesnt need optimisation
expensive

Question 18

How is cDNA library used and screened/why

Answer 18

• prepared in plasmids or phage vectors and must be screened soon after creation because of short half lives

Question 19

How do you preserve a plasmid-based library

Answer 19

• amplify unsegregated library then mix with glycerol, freeze it and store at 80

Question 20

How do you amplify phage based library

Answer 20

• grow a phage infected bacterial culture and harvest the phage after lysis
• store with chloroform at 4

Question 21

How do you perform microarray detection

Answer 21

• cDNA are made from mRNA or total RNA extracted which can be sampled from differe populations or treatment conditions
• If different populations label with different dyes then mixed and hybryised in a humidified chamber agasint same array
• the two population compete for the same target or spot
• visualise array in laser induced fluro with confocal CCD camera detector

Question 22

How is the spot intensity determined

Answer 22

• spot intensity at the two wavelengths is determined and a ratio or log ratio between the
  two fluorescent intensities is calculated.

Question 23

What tools can you use for RNA seq

Answer 23

452 pyrosequencing
Illumina sequencer
third gen SMMRT

Question 24

What is RNA seq also called

Answer 24

whole transcriptome shotgun sequencing

Question 25

How do you make a gene library

Answer 25

• mRNA 3’ (poly(A)) tail is targeted in order to ensure that coding RNA is separated from noncoding RNA.
• This can be accomplished simply with poly (T) oligos covalently attached to magnetic beads.
• Using poly(T) magnetic beads, the flow-through RNA (non-poly(A) RNA) can yield
  important noncoding RNA gene discovery.
• Ribosomal RNA represents over 90% of the RNA within a given cell.
• The next step is reverse transcription.
• The cDNA can be further fragmented to reach the desired fragment length.

Question 26

What are the limitations of RNA seq

Answer 26

• unknown ligation bias
• high sbudance transcripts (like house keeping genes) are responsible for majority of sequencing data
• 5% of genes give rise to 75% of sequenced reads, hard to measure accurate abundances

Question 27

Compare and contrast single cell vs bulk RNA seq

Answer 27

*bulk RNA seq averages expression over millions of cells
* single cell captures sample heterogeneity