L12 - polymerase chain reaction Flashcards
(19 cards)
what is Polymerase chain reaction
PCR
efficient and rapid in vitro enzymatic amplification of specific DNA or RNA
amplifies any DNA sequnce using a template strand between opposing oglionucletide primers
= synthetic short single stranded DNA fragments
why do we use PCR usually
make enough of a target molecule for it to be analysed
what are the requirements/4 basic ingredients for PCR
DNA template
DNA polymerase + buffer + Mg2+
dNTPs
DNA primers
= inside a thermocycler
what direction is DNA synthesis always
5’ to 3’
adding on a 5’ phosphate of a dNTP onto the 3’ hydoxyl group
how many primers are in PCR
2
opposing Forward and Reverse primers = 1 on each strand
DNA pol adds dNTPs to 3’ end of primers = area outside of primers is not amplified
what is the basic proscess of PCR - describe the first cycle
- heat denaturation:
94℃ for 30s = seperates strands - Annealing:
different temp depending on primers used for 30s = allows primers to bind to open strands - Extension:
72℃ or 68℃ for however long needed = depends on length of sequence were trying to amplify. Temp also depends on the polymerase used
why do we get intermediate products in PCR forcing us to continue the cycles
we only want the area of the primer and then the following area
we can get intermediates due to the area after the rpimers still being present
= when PCR happenes again the strand with primer and the copied sewqunce will split and then be copied
= this strand will NOT have the area outside the primer and is our target
= see L12 slide if confused
why does PCR plateau
primer and dNTP depletion
= PCR is exponential so runs out relatively quickly
what does th term ‘high fidelity’ mean
accurate
= rarely amkes mistakes when copying strand
why would we use a low fidelity polymerase in PCR
makes lots of mistakes due to lack of proof-reading
lots of added mutations = perfect for mutagenisis
descibe the optimal primer
specific to a sequnce with high binding affinity
18-24 bases long = specific enough sequence
melt temp of 55-65℃
high G-C content especially at 3’ end of primer for stronger binding = this is the bit that nucletides will be added to
= it needs to be stable and not flop out of position
what is the Tm in terms of PCR
melting temp
temperature that 50% of dsDNA will dispciate into single stranded DNA
= higher the GC content of the DNA the higher the temp required
why should primers avoid using repeated sequences/runs
increases probability of binding to non-specific sequences found in multiple parts ofb the genome
how much lower should theb annesling temp (Ta) be to the melting temp (Tm)
2-5℃ lower
= forwards and backwards primers should also have similar melting temps
3 different applications of PCR
- PCR for cloning:
amplificaytion of a piece of DNA - PCR for gene expression:
reverse transcription PCR (RT-PCR) to amplify a piece of DNA from an mRNA sample
= RT-PCR = semi-quanitative
- PCR for RNA virus detection:
amplifying DNA from an RNA genome
= real time PCR (qPCR) = quantitative
when cloning a gene with PCR we can use Restriction enzymes HOWEVER somtimes there are not restriction sites flanking the desired gene. what can we do to solve this
add restriction sites via PCR
= adding the restrcition site sequences in the 5’ end of the primers does not affect the binding of the 3’ end
how does Reverse transcription PCR (RT-PCR) work to asnalyse levels of gene expression
RT-PCR detects whether a gene is being expressed and then meausres how much mRNA is present to gage how active it is
- isolate mRNA from cell
- convert into cDNA via RT
= produces a DNA strand annealed to mRNA template strand
- remove mRNA with alkali or RNase H
- amplify fragment via PCR
= amount of amplified DNA reflects amount of mRNA in cell and therfore gene expression
how can contamination with genomic DNA in Reverse transcriptase PCR be a problem and how can this be solved
RT-PCR is meant to measure mRNA levels — not DNA!
During PCR, your primers might accidentally bind to genomic DNA from the cell that accidentally got in –> not just your cDNA from the mRNA
That gDNA can be amplified by PCR, giving false positives —> it looks like the gene is being expressed even if it’s not.
= add DNase to cut up any DNA BEFORE reverse transcription
how does Real-time or quantitat8ive PCR work and what for
decetion of RNA viruses in cells
- isolate RNA from a cell = this will inculde viral RNA if the person is infected
- turn into cDNA via Reverse transcriptase
= PCR only works on DNA
- qPCR amplification with sequence-specific flourescently labbeled primers
= Fluorescence increases with each cycle = more DNA = more viral RNA originally
- Real-time detection = the more RNA originally present → the faster fluorescence crosses a threshold (called Ct or Cq value).
= the lower the Ct the less number of cyles to cross thereshold = more original viral RNA
