PCR technique Flashcards
Denaturation
Denaturation is the first step of the reaction. The reaction is initially heated to 95°C. This is to melt
the DNA, separating it into single stranded DNA allowing access for primers to bind. At the start of
the reaction, an extended denaturation of 5 minutes is used to initialise the reaction. After this, the
denaturation step of each cycle is 1 min.
Annealing
where the temperature is reduced to 50-65°C allowing specific
primers to anneal to their specific target sequence on the template DNA. The temperature for this
step varies depending on the primer length, sequence and complexity. In order to estimate the
optimum annealing temperature, we first need to calculate the melting temperature (Tm) for the
primers, that is, the temperature where 50% of primers will dissociate from the DNA template. The annealing
temperature is usually ~5°C below the Tm but sometimes needs to be optimised for the specific
reaction.
Extension
e. This is performed at 72°C, the optimal temperature
for Taq polymerase (see below) activity. This step involves the addition of nucleotide bases (dNTPs)
to new strands and during the cycles last for 1 min. At the end of the reaction, after 30 cycles, a final
extension step is included for 8 minutes to ensure all newly synthesised strands are fully elongated.
Taq Polymerase
A DNA polymerase is responsible for catalysing the addition of nucleotides to the new strand of
DNA, complementary to the template strand
Taq polymerase, a commonly used, thermostable DNA polymerase taken from a thermophilic bacterium.
What happens when there’s insufficient taq polymerase?
Insufficient Taq polymerase will result in reduced PCR
product replication.
DNA Template
The template is the original sample DNA that is to be amplified. The template contains the target
sequence that primers will anneal to. PCR can amplify very small amounts of DNA
Too little DNA Template
too little starting material, the reaction will not amplify enough PCR product. In some cases, this can
be prevented by increasing the number of cycles.
Too much DNA Template
Too much DNA can hinder the reaction by
sequestering Mg2+ or exhausting primer concentrations. For a successful PCR reaction, it
is critical that the DNA template is of good quality, free from contaminants.
Magnesium Chloride (MgCl2)
Magnesium is a co-factor for Taq polymerase and is thus essential for its activity. Insufficient
MgCl2 will result in reduced or no PCR product while too much MgCl2 can cause non-specific
primer-binding or reduced dsDNA denaturing.
Primers
Primers are short pieces of DNA that are complementary to very specific sections of the template
DNA. During the annealing step of the PCR reaction, primers bind to their complementary targets
and provide Taq polymerase a starting point for synthesis. Primers are generally custom made based
on the known sequence of the target to be amplified. DNA polymerase can only synthesise new
strands of DNA in one direction. Therefore, 2 different primers are required: one to bind to the 3’
end of each template strand of DNA
Excess Primer
If excess primer is present, non-specific binding to non-target sites of the DNA can occur. This will
result in amplification of the incorrect section of DNA. Increased primer concentration may also
cause primers to bind to each other, forming what we call primer dimers.
Too little primer
If primer levels are too
low, there will be a reduced annealing efficiency and ultimately less PCR product.
dNTPs
These are the nucleotide bases that are used to generate new strands: A, T, C and G.
Insufficient dNTPs
Without sufficient concentration of dNTPs, there will be insufficient material to synthesize the new DNA
Too much dNTPs
. If dNTP concentrations are too high, Mg2+ may be depleted as dNTPs can sequester Mg2+ ,reducing
the concentration of free Mg2+ for enzymatic activity.
