PCR Flashcards
What is PCR?
- polymerase chain reaction
- uses polymerase enzyme to read a single strand of DNA and extract info from nucleotides to build new DNA
- it involves scaling up strands of DNA so there is enough material to do analysis
Why is PCR needed in forensic science?
- only got few pg in what we are collecting as forensic scientists
- no techniques to analyse such a small amount of DNA so need to be method to make identical copies to get enough to be able to analyse it
What are the steps involved in PCR?
Why is temperature changes important?
1 - start with double stranded template of DNA
2 - heat up to 94 C to denature and split two strands to get two single helixes
3 - hybridise primers at 64 C (add primers to one end of DNA (3’ to 5’ end))
4 - use polymerase starting on primer to attach nucleotides (complementary bases) from the environment to template strand to artificially replace DNA at 72 C
5 - now got two double stranded DNA repeats that are exact copies of starting template
6 - repeat
- temperatures change throughout process as are optimised to be able to cycle with all reactants
What can we use PCR for?
- identify entire genes in less than a week
- detect mRNAs and look at gene expressions
- identify hereditary diseases (and how to tackle them)
- identify viruses (COVID-19) or microbes
- site-directed mutagenesis (when things become mutant/changed in environment)
- paleobiology (looking at fossils and understanding ancient DNA of things)
- useful for environmental trace evidence (diatoms, forams) – genetic profiling of these is incredible useful as allows us to identify locations
- can create library to identify these a bit better
- identify familial relationships e.g. parental testing
- in forensic investigations
What are four or five core PCR components?
- template
- primers
- taq polymerase
- dNTPs
- buffers and salts
What is role of template in PCR?
- what it is
- how much is needed
- does it need to be pure
- what we want to replicate
- it is the sample we have collected
- only need a few pg but ng is preferable as at pg level background DNA is issue
- selective amplification means purity isn’t crucial but helps
- if noisy/messy/dirty sample we can get around this
What is role of primers in PCR?
- why it is needed
- importance of primer sequence
- what primers do
- what must we ensure primers allow for
- what does primer concentration determine
- polymerase needs primers because it can only add bases to pre-existing strands
- primer sequence needs careful design to ensure proper binding to right site only
- primers are made by solid phase phosphoramidite chemistry (gives control to design and modify our primers to our needs)
- primers tell polymerase where to start and stop copying (put where want to start and put where want to end)
- primers are designed to target edge of short tandem repeats (STRs) of DNA which are highly variable between people
- number of alleles within and length of STR is variable between people
- must analyse enough STRs to give a statistically unique DNA profile
- primer concentration determines the maximum yield of product
- primers are used up in each cycle
- more primer = more product
What is role of Taq polymerase in PCR?
- properties
- what it does
- efficiency
- taq polymerase is heat resistant version of polymerase enzyme
- it is stable for short periods of time at more than 90 C so stable in process
(9 mins at 98 C and 30 mins-120 mins at 90 C) - binds to the template strand at the start of area for amplification
- polymerase synthesises new DNA strand by extending primers along amplification area
- it is reasonably efficient - per taq polymerase enzyme can replicate a 1000 base pair strand in 10 seconds
What is role of dNTPs in PCR?
- role
- structure
- what are four different ones
- how much used
- how do they work
- Deoxynucleoside 5’triphosphate (dNTPs) - building blocks of new DNA
- genetic base pairs attached to sugar which is attached to triphosphate
- dATP
- dCTP
- dGTP
- dTTP
- 200 uM of each is added to allow the Taq polymerase to build
- polymerase likes dNTPs, takes them in and builds them up
- builds of 3’ end of DNA
- OH group on 3’ end of DNA allows bridging to next base pair via the phosphate group
What is role of buffers and salts in PCR?
- role of buffer and common example
- why salts needed
- example of salt and its role
- without salt and buffer
- what is needed for
- all must be in buffer e.g. Tris-HCl
- supply correct pH and have additives (magnesium, potassium) to help optimise DNA denaturing, renaturing and polymerase activity
- need salts to stabilise everything
- MgCl2 - 0.5 - 4 uM Mg2+
- starting blocks have massive negatively charged phosphate groups
- to ignore ES repulsion, need enough Mg2+ to bridge across and stop ES repulsion
- without salt and buffer would denature
- needed for hybridisation and extension
What are 7 steps of PCR process?
1 - initialisation
2 - denaturation
3 - annealing
4 - extensions
5 - cycling
6 - final extension
7 - final hold
What happens during initialisation stage of PCR?
- 94 – 96°C for 30s – 5 minutes
- ensures template DNA is fully suspended and properly denatured (especially important if the template is very long)
- hot to activate hot start polymerases
What happens during denaturation stage of PCR?
- 94 – 98°C for 30s
- splits double stranded DNA into single stranded DNA
- ensure equilibrium is pushed (higher temp) so makes DNA more stable in single strands rather than double helix
What happens in annealing stage of PCR process?
- 50 - 64°C for 30s
- binds primers to template strands
- temperature must be a few degrees lower than Tm of primers
- primers bind over complementary templates because of high concentration
- polymerase will also bind at this stage but not proceed
What happens during extension stage of PCR process?
- 72°C for 30s (or 1 minute per 1000 base pairs)
- polymerase synthesises new complementary DNA strand by extending primers along whole amplification area
- polymerase adds free dNTPs from reaction mixture to template DNA
- temperature is optimised for activity of the Taq polymerase (enzyme) (If lower temp, enzyme won’t work as effectively)
What happens during cycling stage of PCR process?
- 15 - 40 Repeats of denaturation, annealing and extension.
- each cycle doubles DNA concentration
- too few = not enough amplification
- too many = limited by dNTP concentration (leads to truncated products as hasn’t gone to completion)
What happens during final extension stage of PCR process?
- hold at 72°C for Several Minutes
- ensures all strands are finished
- reduces truncated products
What happens during final hold of PCR process?
- 4°C until needed (most stable)
- best condition for storing product as most stable temp so DNA will not degrade
What is PCR instrumentation like?
- when being developed, used to be massive
- machine is now smaller - does all the cycling for us
- has wells for us to put sample tubes in
- program the temperature sequence - fully automated
What is a primer dimer and how is it formed?
- primer binds together rather than onto the strands and get double helix again
- occurs due to:
- poor choice of primer sequence
- too much primer added (primer conc defines progress of reaction)
What three issues can we get with PCR?
- no amplification
- non-specific amplification
- weak amplification
No amplification:
- what does this mean
- 5 reasons why
- can see no bands on gel
1 - primer too concentrated (get primer dimers)
2 - dNTPs degraded by freezing (dNTPs feedstocks for polymerase to go in)
3 - template has degraded (poor storage of sample between collection/lab)
4 - annealing temp too high
5 - you forgot to add something
Non-specific amplification:
- what does this mean
- 2 reasons why
- lots of random bands/blurred bands
1 - contamination - something else is in there interacting with dNTPs, polymerase etc.
2 - annealing temperature too high
Weak amplification:
- 3 reasons why
1 - concentration of any PCR components is too low (not enough material in order to actually build up and create repeat structures e.g. not enough primer
2 - not enough cycles (hasn’t been amplified enough)
3 - annealing time is too short (DNA still in double helix)
Reverse transcription PCR
- process
- what is it more aimed towards
- RNA to DNA:
- bind primer (primer designed to interact with RNA instead of DNA) to RNA
- undergo reverse transcription
- then denature to form cDNA
- bind primer to cDNA
- then PCR to make copies of cDNA
- more for bioscience applications
- no immediate application in forensic science
Quantitative PCR
- what info does this give us
- graph?
- what is process?
- allows us to know how much is being expressed through qPCR
- graph shows number of cycles vs fluorescence response
- as number of cycles increases, fluorescence response increases
- after 30-40 cycles it plateaus (run out of material or amplification isn’t going to add any more info)
- know when to stop
- add fluorescence molecule into PCR process (need PCR machine that has fluorescent detector and can track progress as building number of repeats)
- make conc of DNA product proportional to fluorescence
- add primer (containing fluorophore (fluorescent tag) and quencher (stops fluorescence))
- when primer is on template and untouched it is bound to single strand of DNA it has a fluorophore and wants to fluoresce but is immediately quenched as quencher too close to fluorophore
- as polymerase moves along, digests primer, this removes fluorophore and quencher so fluorescence will happen
- amount of fluorescence in solution is directly related to number been released
What mode of PCR was used for SARS-CoV-2 testing?
- process
- how did results work
- reverse transcription quantitative polymerase chain reaction
- swab nose and throat
- mix with extraction buffer (breaking out DNA from cellular stuff in saliva/snot)
- will be RNA so do reverse transcription approach
- then do qPCR
- allows us to identify viral load quickly
- picked primers to highlight base pairs specific to SARS-CoV-2 but not SARS-CoV
- tells us if sample positive or negative
What is digital PCR?
- method
- why would we want to do this?
- doing qPCR on a larger scale (across as many wells as we can fit in PCR machine)
- more sensitive measurement of amount
- gives more precise read as doing multiple repeats
Considering PCR, why do the two template strands get bound by primers after annealing instead of binding each other? [4 marks]
- temperature of annealing is above Tm of DNA but a few degrees lower than Tm of primers, as a consequence binding of a DNA strand to a primer is thermodynamically favoured over forming a duplex
- concentration is also an important factor here, need sufficient primer to drive DNA-primer reaction forward, but not so much that primer dimers are formed.
