Application of PCR and nucleic acid hybridisation Flashcards
What are primers for PCR designed to be?
Complementary to nucleotide sequences of interest within a genome
How do primers bind to a sequence of interest in PCR?
Sequence complementarity
How can RNA be analysed using RT PCR?
Reverse transcriptase is used to make a DNA copy of the RNA
What is needed to be done to mRNA before PCR is done?
A primer that binds to the polyA tail is added
Why is a primer that binds to the polyA tail added to the mRNA?
To allow the rest of the nucleotides to bind
What do some DNA polymerases have that is important for real time PCR?
Exonuclease activity
What does the exonuclease activity allow DNA polymerases to do during real time PCR?
Allows them to degrade anything downstream (e.g. an oligonucleotide)
Where does the probe sit in real time PCR?
Between the forward and reverse primer
What is the probe labelle with?
A reporter group and a quencher group
What is the role of the reporter group?
To be fluorescent dye
What is the role of the quencher group?
To “quench” the fluorescence from the reporter group, so when they are near each other no fluorescence is detected
What does the 5’ to 3’ exonuclease activity do as the extension proceeds?
Causes the probe to degrade as it will be downstream
What is the outcome of the probe degrading?
The reporter group and quencher group will no longer be in close proximity to each other
What happens as a result of the reporter group and quencher group not being in close proximity?
The fluorescence from the reporter group will be visible
What can be said about the initiation phase if the starting material is in low number?
Longer initiation phase
What can be said about the exponential phase if there is a lower amount of starting material?
It will rise later
WHat is DNA partitioned into in digital PCR?
Microreactors
How many microreactors are there relative to DNA in digital PCR?
Vastly more to make it likely that only one molecule enters each microreactor
What happens once the DNA is in the microreactors?
The PCR components are added, and PCR is run
What are the microreactors?
Water droplets in oil
How are the number of PCR reactions counted in digital PCR?
It is labelled fluorescently, and a laser scanner is used to count the number of positive PCR reactions
Why is PCR necessary for to count the number of each DNA fragment?
There is too little DNA without it, so the fluorescence level would be too low
How can digital PCR be used to detect rare alleles?
The compartmentalisation of each PCR reaction per DNA fragment means that when they are all amplified they amplify independently–> ones with less won’t be swamped
First step of nucleic acid hybridisation?
Oligonucleotide probe of interest is hybridised to nucleic acids that contain test or reference samples
What are the nucleic acids that the probe is hybridised to fixed to?
A solid support
Second step of nucleic acid hybridisation?
Nucleic acids, solid support and oligonucleotide probes are washed to ensure specific binding
What is in situ hybridisation used to visualise?
The patterns of mRNA transcript localisation in developing embryos, tissue sections and cell cultures
What is done to the tissue of interest in In Situ Hybridisation?
Embedded in paraffin, and thin slices are cut
Slices are then fixed onto a glass slide, and nucleic acid hybridisation is performed on them
What is used to bind the oligonucleotides in a microarray?
A microarray–> silicone chip
What are the oligonucleotides in a microarray complementary to?
Each protein coding gene in the human genome
How does a microarray work?
The DNA fragments that you add will bind to the oligonucleotide that is complementary to them–> stronger signal for more binding = more copies present in sample
What must be done to mRNA from two different samples of the same tissue type before a microarray can be done to compare gene expression in them?
They must each be labelled with a different colour
What happens in a comparative microarray if no colour is present for a gene?
Neither sample has expressed that gene
What is known in a comparative microarray if the colour present on the scan is a mixture of both colours?
Both samples have the mRNA for that gene present
What happens if only one colour is present on the scan of a particular gene for a microarray?
Only one of the samples has the mRNA for that gene
