Genetics - Gene Expression Analysis Flashcards
What is reverse transcriptase analysis based on?
Some organisms contain a high abundance of a specicif mRNA like chicken oviducts ovalbumin
Reverse Transcriptase
An enzyme transcribing ssRNA into DNA, able to synthesised a double helix DNA once the RNA has been reverse transcribed
Complementary cDNA
A DNA synthesised from an ssRNA template catalysed by reverse transcriptase
How is DNA cloned with RT?
Uses ssRNA to synthsise a complementary strand of DNA
What is the stem loop structure in RT?
Where the DNA folds back on itself at the 3’ end, consisting of a short dsDNA unit
What is the advantage of cDNA?
Does not contain the introns foudn in genomic DNA
What is done with cDNA?
Amplified by PCR then Joined to a vector.
RT-PCR
This is conversion of RNA to cDNA by RT with then PCR of the cDNA
What can RT-PCR be used for?
Comparison of gene expression between samples(different embryonic stages, tissues or same cell under diff ocnditons)
What is the first step of ligating cDNA to a vector?
Vector must be cleaved with RE and linearised, creating a site of which cDNA can be inserted.
How is the cDNA then generated?
From mRNA using RT, where adaptors are modified to both ends containing specific RE sites.
How are complementary overhangs of both vector and cDNA generated?
Using the same restriction endonucleases
What is the function of DNA ligase?
Covalently binds DNA strands of 5’ phosphate of one to 3’ hydroxyl of the other.
What is phosphodiester bond formation coupled with?
Pyrophosphate hyrdolysis with negative G change.
What is the structure of DNA ligase?
NTD recognises DNA subsrate
Central catalytic domain catalyses bond formation
CTD interacts with proteins and cofactors.
How is RNA isolated for RT-PCR?
Phenol-chloroform extraction, Column-based RNA purification or magnetic bead-based isolation.
What is the process of RT-PCR?
PCR amplified where product analysed by fluoressence or real-time PCR(qPCR)
How is RT-PCR gene expression analysed?
If amount of PCR product is lower in experimental group, the gene has lower expression, as amount of RNA transcript is generally correlated with level of gene expression.
What is the process of microarray expression analysis?
cDNA generation, labelling with Cy3/Cy5, hybridization with primers on microarray where fluorescence signals intensity analysed.
Photolithography
This is where light is used selectively to remove material from a substrate
How is a microarray chip generated?
Photolithography.
Why is DNA microarray advantageous?
Analysis of expression of thousands of genese at one time using DNA microarray.
What is the structure of a DNA microarray chip?
A microscope slide printerd with thousands of slots in defined positions, each containing a known DNA sequence.
How is microarray analysis performed?
mRNA collected from a reference(healthy indiviudal) and experimental(a diseased individual)
Both labelled with unique fluorophores after conversion into cDNA
Hybridised with microarray slides where emissivity is assesed.
In Situ Hybridization
This is a technique used to detect/localise speciifc NA sequences within intact cells or tissues.
What is the first step in ISH?
Probe prepared being a DNA/RNA complementary to target sequence of interest, labbeled.
What is the second step in ISH?
Tissue of interest fixated and inserted with the probe, hybridizing within cellls or tissue with complementary sequence.
What is the third step in ISH?
Bound probe is detected using fluorescent methods and visualised under confocal microscopes to visualise location of target NA.
What can ISH be used for?
Locating where single genes are expressed as well as time and location of the mRNA in the sample.
How is ISH visualised?
Auroradiography?
Autoradiography
This is the detection/visualisation of radioactive molecules by emitted radiation in contact with photographic fillm.
How does Autoradiography work?
Silver halite crystals are excited and destabilised, attracting silver ions from solution forming clusters.
What are the clusters in AR proportional to?
Amount of radiation.
What does northern blotting use instead?
RNA in formaldehyde in agarose gel in GE with transfer to membrane.
Epigenetics
This is the study of heritable changes in genome function not assocaited with DNA sequqence alteraitons.
How is epigenetic regulation determined?
3rd Generation Seqeuncing
Why is Epigenetic modificatio nimportant to determine?
A bacteria might gain advantages by epigenetic regulation isntead of genetic.
