L7: Gene Expression and Gene Expression Profiling Flashcards
Central Dogma
DNA => RNA => Protein
How do you turn genes on and off
controlling transcription
Define promoter and operator
promoter: DNA segment allowing gene to be transcribed
operator: part of DNA that turns gene on and off
What does an operon include
operator
promoter
one or more structural gene
Where are operons most common
prokaryotes
What does a lac operon contain
three genes encoding for enzyme that break down lactose
on and off of lac operon
off : no lactose, has repressor
on: lactose present
What is transcription controlled by
regulatory DNA sequences and protein transcription factors
TATA box
enchancer + silencer
regulatory sequences
What else is important in gene regulation
RNA processing
What is a transcriptome
set of transcropts and relative expression levels
what is the objective of gene expression profiling
moniter mRNA abundance and changes to understand gene function
List 6 methods of gene profiling
- northern blotting
- RT PCR
- cDNA
- Microarray
- Bulk RNA seq
- Single-cell RNA seq
What are two mechanisms of microarrays
hybridisation by cDNA spotted array or oligonucleotide array
What is Bulk RNA seq
sequencing based on gene expression profiling
Taq polyermase only works with DNA so how do we use it
reverse transcritpn RNA => cDNA
How does SYBR green work, why use it, how
binds to ds DNA and emits like when illuminated at a wavelength
why: dsDNA intercalating dye, unspecific = optimal = cheap
how: apply => extend =>apply excitation
What is taqMan probe chemistry
sequence specific
doesnt need optimisation
expensive
How is cDNA library used and screened/why
- prepared in plasmids or phage vectors and must be screened soon after creation because of short half lives
How do you preserve a plasmid-based library
- amplify unsegregated library then mix with glycerol, freeze it and store at 80
How do you amplify phage based library
- grow a phage infected bacterial culture and harvest the phage after lysis
- store with chloroform at 4
How do you perform microarray detection
- 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
How is the spot intensity determined
- spot intensity at the two wavelengths is determined and a ratio or log ratio between the
two fluorescent intensities is calculated.
What tools can you use for RNA seq
452 pyrosequencing
Illumina sequencer
third gen SMMRT
What is RNA seq also called
whole transcriptome shotgun sequencing
How do you make a gene library
- 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.
What are the limitations of RNA seq
- 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
Compare and contrast single cell vs bulk RNA seq
*bulk RNA seq averages expression over millions of cells
* single cell captures sample heterogeneity
