Lecture 5 - PCR Flashcards
what is the basic principle of the PCR reaction
polymerase chain reaction
a few pg of DNA is replicated/amplified many times over so we have enough DNA to analyse
we use this to quickly scale up a small amount of DNA to give us enough to analyse
What are the components needed for a PCR reaction (5)
- template strand
- primers
- taq polymerase
- dNTPs
- buffers and salts
what enzyme is used in PCR to aid with replication
Taq polymerase
what are the steps in the PCR process (7)
- start with one double stranded DNA - initialisation
- denaturation
- the primers hybridise
- annealing
- extension - end with 2 double stranded DNA strands that are genetically the same
- repeat = cycling
- final extension and hold
what temp is normally required to denature a double stranded template DNA
94-98 degrees - a high temp to break the hydrogen bonds so the strands can separate
what temp range is required to hybridise the primers
50 - 64 degrees
what temp is required for elongation and what enzyme is involved at this step
72 degrees
DNA polymerase builds 5’ to 3’ taking nucleotides from the surrounding solution
how many PCR repeats equates to 1 billion copies of DNA
30
generally 30-32 cycles are used in PCR
2^30 =1 billion
what are the two biggest commercial uses of PCR
familial relationship testing
forensic investigation
what are 6 things we can do using PCR
look at hereditary diseases
use for gene expression
identify genes quickly
identify microbes or viruses
paleobiology
site directed mutagenesis
why is pure DNA not crucial for PCR
PCR allows for selective amplification due to the use of primers to show polymerase where to begin replication
so purity of the DNA isn’t crucial but it helps
why may only a pg amount of DNA be an issue in PCR
at pg level there may be interference of background levels/noise from instrumentation therefore ng is preferred if possible
what do primers do in PCR
primers are designed to attach to the edge of areas on DNA called STRs (Short tandem repeats) as the length of these are highly variable in humans
we analyse enough STR regions on the DNA to be able to get a profile representative of the individual
these primers signal to DNA polymerase where to start and stop replicating as polymerase can only add bases onto a pre existing strand
we have a stop and start primer for each STR regions considered
how are primers made for PCR
what does this process allow
primers can be synthesised using solid-phase phosphoramidite chemistry
this allows us to control what regions on the DNA are amplified and design/modify the primers to fit our needs
how many bases long are primers used in PCR
tend to be 15-30 bases long
why does the synthesised primer sequence need to be carefully designed
to ensure they bind properly to the correct region on the template DNA strand
what part of the PCR process do analysts have the most control over
the primer designed and therefore what regions of the DNA are being targeted
what does the primer concentration in the reaction mixture determine
the maximum yield of the intended product
the more primers the more likely they are to attach to the DNA
why is Taq polymerase used in PCR (2 reasons)
- this is the heat resistant form of polymerase enzyme - this enzyme is stable at 90 degrees or greater for short periods of time (minutes), other forms would denature (so stable to be used here)
- this enzyme works quickly - can replicate a 1000 base pair strand in under 10s
what is the source of taq polymerase
thermus aquaticus - a bacterium that lives in hot temps
what are dNTPs and what are they used for in PCR
dNTPs = deoxynucleoside 5’-triphosphates
these are the building blocks of the new DNA - building onto the 3’ end of the previous one
how many dNTPs are there and what are they called
4
dATP
dCTP
dGTP
dTTP
how much of each dNTP is added to the PCR reaction mixture
200 microM
what is the purpose of the buffer and salts in the PCR reaction mixture
needed for the stabilisation of the hybridisation and extension processes
the strands are likely to denature without these present
salts - prevent electrostatic repulsion of phosphates
buffers - maintain stable pH
what is meant by hybridisation in DNA
the process where two complementary single stranded DNA bond to become a double stranded molecule
give an example of a buffer and a salt to be added to a PCR reaction mixture
tris-HCl = buffer
salt = MgCl2 (magnesium chloride)
what happens in the initialisation stage of PCR
what is the temp and time normally here
ensure the template DNA is suspended in solution and begins to denature
some ‘hot start’ polymerases are activated here
94-96 degrees for 5 min
when is the initialisation stage particularly important
when we have a very long template DNA strand
longer strands may need higher temps or longer initialisation times
what happens in the denaturation stage of PCR
what temp and time are used here
the double stranded template DNA is split into two single strands
94-98 degrees for 30 secs
what happens in the annealing stage of PCR
what temp and time are used here
what does the temp need to be lower than
primers bind to template strand
polymerase binds but doesn’t proceed
50-64 degrees for 30 secs
a few degrees lower than the melting temperature of the primers
what happens in the extension stage of PCR
what temp and time are used here
the complimentary strand is synthesised via base pairing - the temp is optimised for the activity of the enzyme
72 degrees for 30 sec - or 1 min per 1000 bases on template
what happens in the cycling stage of PCR
the denaturation, annealing and extension are repeated
can be anywhere between 15-40 repeats
every cycle doubles the conc of DNA
what can too many PCR cycles lead to
limited dNTP concertation in solution so the strand is not synthesised correctly
can lead to truncated products = partial fragments of the intended products
what can too few PCR cycles lead to
not enough amplification has occurred so the regions you want to analyse may not be able to be analysed
what happens in the final extension stage of PCR
what temp and time are used here
ensures all the strands have finished being replicated and reduce the risk of having truncated products
72 degrees for a few minutes
what happens in the final hold stage of PCR
what temp and time are used here
best storage condition for the PCR products
4 degrees until you use the products in electrophoresis
what do the primers define in PCR
the length of the STR region being amplified
how do PCR instruments work
Eppendorf sample tubes are places into the instrument
can do multiple samples at once
then programme the temperature sequence to what you need
what is a common problem seen in PCR and what causes this issue (2 reasons)
the formation of primer dimers
due to the poor choice of the primer sequence or if too high a conc of primer is added
what is a primer dimer
where two primers join to each other rather than onto the template DNA
when do primer dimers become more of a problem
when looking at multiplexes - analysing more than 1 STR region at once
this is often the case for multiplexes nowadays
give three other problems that may be seen in PCR (briefly explain why each may occur)
1 = 5 reasons
2 = 2 reasons
3 = 3 reasons
- no amplification of the DNA
- too high primer conc
- dNTPs degraded by freezing
- degraded template strand
- annealing temp too high
- human error - forgot to add something to reaction mix - non-specific amplification of the DNA
- contamination
- annealing temp too high - weak amplification of the DNA
- DNA conc too low
- not enough PCR cycles
- annealing time too short
what is RNA used for and how can it be used in DNA replication
DNA give info on what cell is capable of whereas RNA gives info on what the cell is doing and builds proteins we use
we ca do reverse transcription to get DNA sequences from RNA ones:
- primers can be designed to bind to the RNA,
- synthesise the DNA strand from this
- denature from the RNA strand and then go on to be used in PCR
normally used in biosciences not forensics
what is qPCR
quantitative PCR
a method that measures the concentration of the DNA product
used to track the process of the PCR cycles
how is the concentration of DNA products measured in qPCR
the conc is proportional to the fluorescence signal
detects the fluorescence in real time
how do DNA molecules being replicated on PCR show fluorescence for it to be detected in qPCR
a probe is attached to the template DNA strand
the probe has a fluorophore and a quencher attached to it
the quencher turns off the fluorescence, as polymerase goes along the template strand when it reaches the probe it digests it so the fluroescence is released into the solution and will now give a signal
what does qPCR allow
the progress of the reaction to be tracked so any problems in amplification can be identified early on so we don’t waste time do all the cycles to realise it didn’t work
in qPCR how many PCR cycles does it take for us to see a plateau in the DNA product concentration
why is this plateau seen
30-40 cycles - never do more than 40
due to running out of dNTPs to synthesise new strands or other components in the reaction mixture e.g primers
what 6 components would be added to the PCR reaction mix to develop the covid vaccine
as vaccines use RNA the reaction mix is different to that used in DNA analysis of crime scene samples
reverse transcriptase
dNTPs
primers - these picked to highlight regions we want
taq polymerase
probe strands
what is dPCR
digital PCR
scales up qPCR over multiple wells - qPCR = normally 1 sample at a time
use multiple wells and then count how many show amplification
why might dPCR be used
more sensitive than qPCR and more precise measurement of DNA concentration
