Polymerase Chain Reaction

Question 1

What is polymerase?

Answer 1

• Polymerase is an enzyme that is most involved in the building and repairing in DNA
• It goes along and identifies the end of damaged DNA (3’ end) and attaches and takes some of the excess nucleotides and enzymatically attaches them along the gap until it is sealed.

Question 2

Polymerase chain reaction

Answer 2

• Uses polymerase to use its ability to read a single chain of DNA and extract things from the environment (nucleotides) and build new DNA.
• We don’t have techniques that allow us to analyse such small amounts (pg) of DNA
• We need to scale it up so we have enough copies that we can analyse.

Question 3

Polymerase chain reaction process

Indepth

Answer 3

• Start with double stranded template DNA that we want to reproduce
• we first denature it (split the two strands)
• This results in two single helices sitting in solution which is done by heating it
• We than have primers that stick onto one end of the DNA
• We then add polymerase that sits on the 3’ to 5’ end and builds on the 5’ to 3’
• Polymerase is good at polymerasing along the chain
• It takes nucleotides from solution and making a match to the other side
• It looks at the base on the other side and finding the pair to attach
• It replicates the opposite base pair artificially
• We get two double strands of DNA which are exact copies of each other and starting material
• This process is repeated again and again until we have enough of the starting material.

Question 4

Polymerase chain reaction process

basic steps

Answer 4

1. Double stranded template DNA
2. Denature it @ 94oC to split it
3. Hybridise primers @ 64oC
4. Elongation @ 72oC
5. We end up with 2 double stranded DNA
6. Repeat until there is enough starting material

Question 5

Impact of polymerase chain reaction

Answer 5

• Identify genes in <1 week
• Detect mRNAS for gene expression
• Identify hereditary diseases
• Identify viruses or microbes
• Site-directed mutagenesis
• Palebiology
• Identify familar relationships
• Used for forensic investigations to amplify DNA

Question 6

PCR Components

Primers

Answer 6

• Polymerase needs primers because it can only add bases to pre-existing strands.
• Primer sequence needs careful design to ensure proper binding to the right site only - 15-30 bases long.
• Most of human DNA sequences same in every person - DNA Profiling targets repetitive sequences (Short Tandem Repeats - STRs) which are highly variable.
  *

Question 7

How are primers made?

Answer 7

• Primers made by solid-phase phosphoramidite chemistry which gives us control to design and modify our primers to our needs.
• We design primers to target the edge of STRs.
• Analyse enough STRs gives a statistically unique ‘DNA profile’.
• Primer design and sequence is important.
• The length and number of alleles are highly variable in STRs so we target these to give us enough uniquely identifiable material.

Question 8

What do primers tell polymerase?

Answer 8

Primers tell polymerase where to start and stop copying.

Question 9

What does primer concentration determine?

Answer 9

• Primer concentration determines the maxium yield of the produced as they are used up in each cycle.
• In theory more primer leads to a higher yield.
• However, in reality at a certain point the yield plateaus.

Question 10

PCR components

Taq polymerase

Answer 10

• Polymerase would get destroyed if it was heated up to 90oC (which is done to denature the DNA).
• Taq Polymerase is a heat resistant version of the polymerase enzyme.
• Stable for short periods of time at 90°C<.
• Can replicate a 1000 base pair strand of DNA in under 10s!!.

Question 11

PCR componenets

dNTPs

Answer 11

• Deoxynucleoside 5’-triphosphates (dNTPs)
• Polymerase takes them and uses them as building blocks for new DNA.
• 200uM of each added to allow the Taq Polymerase to ‘build’.
• Build off the 3’ end of the DNA
• Bases attached to a sugar and triphosphate

Question 12

PCR Components

Buffers and salt

Answer 12

• Buffer to stabilise it
• Normally a Tris-HCl buffer
• The salt is there to stabilise DNA due to negative phosphate groups
• Without the buffer it would denature.
• Needed for hybridisation and extension

Question 13

PCR Process

Initialisation

Answer 13

• 94 – 96°C for 30s - 5 minutes
• Ensures template DNA is fully suspended and properly denatured.
• Especially important if the template is
  very long.
• Some ‘hot start’ polymerases require activation to begin working this way/

Question 14

PCR Process

Denaturation

Answer 14

• 94 – 98°C for 30s
• Splits double stranded DNA into single stranded DNA.

Question 15

PCR Process

Annealing

Answer 15

• 50 - 64°C for 30s
• Binds primers to template strands.
• Temperature must be a few degrees lower than T_m of primers.
• Primers bind over complementary templates because of high concentration.
• Polymerase will also bind but not proceed.

Question 16

PCR Process

Extension

Answer 16

• 72°C for 30s or 1 minute per 1000 base pairs.
• Synthesise the complementary strand.
• Temperature is optimised for activity of the polymerase.

Question 17

PCR Process

Cycling

Answer 17

• 15 - 40 repeats
• Each cycle doubles DNA concentration.
• Thirty Repeats = 2³⁰ so copies Made = ~1 Billion
• Too few = Not enough amplification
• Too many = Limited by dNTP concentration
• Too much amplification might result in some other structures within there and get truncated products as it can’t go to completion.

Question 18

PCR Process

Final extension

Answer 18

• 72°C for Several Minutes
• Ensures all strands are finished
• Reduces truncared products

Question 19

PCR Process

Final hold

Answer 19

• 4°C until needed
• Best condition for storing product.
• 4oC is the most stable for DNA, nothing will degrade and it can be stored until we need it.

Question 20

Troubleshooting

Answer 20

• Occurs due to poor choice of primer sequence
• Can occur if too much primer has been added.

Question 21

Trouble shooting

No amplification

Answer 21

• Primers too concentrated.
• dNTPs degraded by freezing.
• Template has degraded.
• Annealing temperature too high.
• You forgot something.

Question 22

Troubleshooting

Non-specific amplification

Answer 22

• Contamination
• Annealing temperature too high
• Lots of random or blurred bands indicates non specific amplifcation.

Question 23

Touble shooting

Weak amplification

Answer 23

• Weak = we see some amplification but not much
• Running out of dNTPs could cause this
• Concentration too low
• Not enough cycles
• Annealing time too short

Question 24

Reverse transcription PCR

Answer 24

• Gives information about what a cell is capable of.
• Gives information about what a cell is doing.
• RNA is the tool the builds our proteins
• Design primers to interact with RNA instead and then build a DNA molecule with RNA and go back to normal PCR reaction

Question 25

Quantitative PCR

Answer 25

• Make the concentration of DNA product proportional to fluorescence.
• Need PCR machine that can detect fluorescence in real time.
• Polymerase will go right to the end and digest probe strand on the way.
• We can add a fluorescence molecule in our PCR so we can track the process of how much material we’re building.
• It starts to plateau bc it either runs out of material or there isn’t anymore.
• A polymerase moves along it will digest the primer and remove the fluorophore and quencher.
• This means we now have something fluorescent hanging in the solution
• The amount of fluorophore is directly related to the amount that has been released so we can track how much it has progressed and how well.

Question 26

Digital PCR

Answer 26

• Many wells
• Count how many wells show amplification
• More sensitive measurement of amount
• dPCR gives a more precise measurements
• qPCR is the same as dCPR but on a larger scale.