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FSCI6370 - DNA > PCR > Flashcards

PCR Flashcards

1
Q

What is PCR?

A
  • 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
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2
Q

Why is PCR needed in forensic science?

A
  • 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
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3
Q

What are the steps involved in PCR?

Why is temperature changes important?

A

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
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4
Q

What can we use PCR for?

A
  • 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
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5
Q

What are four or five core PCR components?

A
  • template
  • primers
  • taq polymerase
  • dNTPs
  • buffers and salts
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6
Q

What is role of template in PCR?
- what it is
- how much is needed
- does it need to be pure

A
  • 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
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7
Q

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

A
  • 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
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8
Q

What is role of Taq polymerase in PCR?
- properties
- what it does
- efficiency

A
  • 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
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9
Q

What is role of dNTPs in PCR?
- role
- structure
- what are four different ones
- how much used
- how do they work

A
  • 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
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10
Q

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

A
  • 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
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11
Q

What are 7 steps of PCR process?

A

1 - initialisation
2 - denaturation
3 - annealing
4 - extensions
5 - cycling
6 - final extension
7 - final hold

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12
Q

What happens during initialisation stage of PCR?

A
  • 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
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13
Q

What happens during denaturation stage of PCR?

A
  • 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
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14
Q

What happens in annealing stage of PCR process?

A
  • 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
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15
Q

What happens during extension stage of PCR process?

A
  • 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)
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16
Q

What happens during cycling stage of PCR process?

A
  • 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)
17
Q

What happens during final extension stage of PCR process?

A
  • hold at 72°C for Several Minutes
  • ensures all strands are finished
  • reduces truncated products
18
Q

What happens during final hold of PCR process?

A
  • 4°C until needed (most stable)
  • best condition for storing product as most stable temp so DNA will not degrade
19
Q

What is PCR instrumentation like?

A
  • 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
20
Q

What is a primer dimer and how is it formed?

A
  • 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)
21
Q

What three issues can we get with PCR?

A
  • no amplification
  • non-specific amplification
  • weak amplification
22
Q

No amplification:
- what does this mean
- 5 reasons why

A
  • 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

23
Q

Non-specific amplification:
- what does this mean
- 2 reasons why

A
  • lots of random bands/blurred bands

1 - contamination - something else is in there interacting with dNTPs, polymerase etc.
2 - annealing temperature too high

24
Q

Weak amplification:
- 3 reasons why

A

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)

25
Q

Reverse transcription PCR
- process
- what is it more aimed towards

A
  • 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
26
Q

Quantitative PCR
- what info does this give us
- graph?
- what is process?

A
  • 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
27
Q

What mode of PCR was used for SARS-CoV-2 testing?
- process
- how did results work

A
  • 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
28
Q

What is digital PCR?
- method
- why would we want to do this?

A
  • 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
29
Q

Considering PCR, why do the two template strands get bound by primers after annealing instead of binding each other? [4 marks]

A
  • 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.

FSCI6370 - DNA (16 decks)

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