Gene Expression Flashcards
What are reporter genes
Nucleic acid sequences that code for eaiait assayed proteins.
They are attached to regulatory sequence of gene if interest.
They produce a protein product that can be detected and measured to monitor the activity of gene of interest.
What are some examples of reporter genes
GFP produces fluorescence that can be detected under a microscope
Beta-galactosidase produced protein that cleaves colourless substrate into blue
Luciferase produces enzyme that catalyses a bioluminescent reaction which can be detected through emission of light
Evaluate the use of reporter genes in gene expression
Luciferase is highly sensitive and is used for quantitative analysis as can be easily measured with a luminometer.
However it doesn’t provide information on distribution and needs the addition of luciferin for detection
GFP doesn’t require additional substrates so better for visualisation of gene expression.
But has a longer half life so may affect real time monitoring.
GUS is valuable to monitor gene expression in plant systems and is easily detectable.
Yet is not used for mammalian systems due to its endogenous presence which may interfere with the exogenous expressed GUS
How in situ hybridisation works
Uses labelled nucleic acid probes that are complementary to target DNA or RNA.
These are then visualised under a microscope to identify position of genes and to detect specific DNA or RNA sequences in cells
How does dye based qPCR work
Sybr green binds to ds DNA and during each cycle of PCR the fluorescent is measured
Cost effective (only requires addition of primers)
But detects all dsDNA produced in reaction including non-target and non-template amplification DNA.
How does probe based qPCR work
TaqMan (flurophore + quencher) binds to target DNA and then is hydrolysed during primer extension. This separates the flurophore from the quencher which can be detected.
This means you reduce the effects of amplifying non target DNA but is more expensive.
How micro arrays used to analyse gene expression
RNA is isolated and RT makes it into cDNA. They are then labelled with probes and hybridised onto a microarray (Consists of millions of individual oligonucleotide probes fixed to solid surface)
This is cost effective and robust
But there is a low dynamic range of assay and limited by lots of background noise and number of probes available.
How is RNA seq used in gene expression
RNA is isolated, converted in cDNA. Linkers and adaptors are added and then everything is sequenced using NGS.
More sensitive and accurate and provides information about transcript variants and isoforms but more expensive.
