LECT-1

2
Learning Outcome
After students(LO)
learn this lecture,
students are able to explain:
12.1 PCR (LO 12.1)
12.2 PCR cycles (LO 12.2)
12.3 Template (LO 12.3)
12.4 Primers (LO 12.4)
12.5 Enzymes (LO 12.5)
12.6 PCR optimization (LO 12.6)
12.7 PCR variations (LO 12.7)
 PCR
❑ If a pair of oligonucleotide primers can be designed to be
complementary to a target DNA molecule such that they can
be extended by a DNA polymerase towards each other, then
the region of the template bounded by the primers can be
greatly amplified by carrying out cycles of denaturation, primer
annealing and polymerization.
❑ This process is known as the polymerase chain reaction
(PCR) and it has become an essential tool in molecular
biology as an aid to cloning and gene analysis.
❑ The discovery of thermostable DNA polymerases has made
the steps in the PCR cycle much more convenient.
❑ Its applications are finding their way into many areas of
science

LO 12.1: Students are able to explain PCR

 The PCR cycle
❑ Figure 1 shows how PCR works.
❑ In the first cycle, the target DNA is separated into two strands
by heating to 95°C typically for around 60 seconds.
❑ The temperature is reduced to around 55°C (for about 30
sec) to allow the primers to anneal to the template DNA.
❑ The actual temperature depends on the primer lengths and
sequences.
❑ After annealing, the temperature is increased to 72°C (for
60–90 sec) for optimal polymerization which uses up dNTPs
in the reaction mix and requires Mg2+.
❑ In the first polymerization step, the target is copied from the
primer sites for various distances on each target molecule
until the beginning of cycle 2, when the reaction is heated to
95°C again which denatures the newly synthesized
molecules.
LO 12.2: Students are able to explain the PCR cycle
 The PCR cycle
❑ In the second annealing step, the other primer can bind to the
newly synthesized strand and during polymerization can only
copy till it reaches the end of the first primer.
❑ Thus at the end of cycle 2, some newly synthesized
molecules of the correct length exist, though these are base
paired to variable length molecules.
❑ In subsequent cycles, these soon outnumber the variable
length molecules and increase two-fold with each cycle.
❑ If PCR was 100% efficient, one target molecule would
become 2n after n cycles.
❑ In practice, 20–40 cycles are commonly used.

LO 12.2: Students are able to explain the PCR cycle

The PCR cycle

LO 12.2: Students are able to explain the PCR cycle

 Template
❑ Because of the extreme amplification achievable, it has been
demonstrated that PCR can sometimes amplify as little as one
molecule of starting template.
❑ Therefore, any source of DNA that provides one or more
target molecules can in principle be used as a template for
PCR.
❑ This includes DNA prepared from blood, sperm or any other
tissue, from older forensic specimens, from ancient biological
samples or in the laboratory from bacterial colonies or phage
plaques as well as purified DNA.
❑ Whatever the source of template DNA, PCR can only be
applied if some sequence information is known so that
primers can be designed.

LO 12.3: Students are able to explain template

 Primers
❑ Each one of a pair of PCR primers needs to be about 18–30 nt long and to
have similar G+C content so that they anneal to their complementary
sequences at similar temperatures.
❑ For short oligonucleotides (<25 nt), the annealing temperature (in °C) can be
calculated using the formula: Tm = 2(A+T) + 4(G+C), where Tm is the melting
temperature and the annealing temperature is approximately 3–5°C lower.
❑ The primers are designed to anneal on opposite strands of the target sequence
so that they will be extended towards each other by addition of nucleotides to
their 3′-ends.
❑ Short target sequences amplify more easily, so often this distance is less than
500 bp, but, with optimization, PCR can amplify fragments over 10 kb in length.
❑ If the DNA sequence being amplified is known, then primer design is relatively
easy.
❑ The region to be amplified should be inspected for two suitable sequences of
about 20 nt with a similar G+C content, either side of the region to be amplified
(e.g. the site of mutation in certain cancers).
❑ If the PCR product is to be cloned, it is sensible to include the sequence of
unique restriction enzyme sites within the 5′-ends of the primers.

LO 12.4: Students are able to explain primers

 ❑ If the DNA sequence of the target is not known, for example when trying to
clone a cDNA for a protein for which there is only some limited amino acid
sequence available, then primer design is more difficult.
❑ For this, degenerate primers are designed using the genetic code (see
Topic P1) to work out what DNA sequences would encode the known
amino acid sequence.
❑ For example HisPheProPheMetLys is encoded by the DNA sequence
5′-CAYTTYCCNTTYATGAAR-3′, where Y = pyrimidine, R = purine and N =
any base.
❑ This sequence is 2×2×4×2×2=64-fold degenerate.
❑ Thus, if a mixture of all 64 sequences is made and used as a primer, then
one of these sequences will be correct.
❑ A second primer must be made in a similar way.
❑ If one of the known peptide sequences is the N-terminal sequence, then
the order of the sequences is known and thus the primer directions are
defined.
❑ PCR using degenerate oligonucleotide primers is sometimes called
DOP-PCR (degenerate oligonucleotide primer-PCR).

LO 12.3: Students are able to explain primers

 Enzymes
❑ Thermostable DNA polymerases which have been isolated
and cloned from a number of thermophilic bacteria are used
for PCR.
❑ The most common is Taq polymerase from Thermus
aquaticus.
❑ It survives the denaturation step of 95°C for 1–2 min, having a
half-life of more than 2 h at this temperature.
❑ Because it has no associated 3′ to 5′ proofreading
exonuclease activity, Taq polymerase is known to introduce
errors when it copies DNA – roughly one per 250 nt
polymerized.
❑ For this reason, other thermostable DNA polymerases with
greater accuracy are used for certain applications.

LO 12.5: Students are able to explain enzymes

 PCR optimization
❑ PCR reactions are not usually 100% efficient, even when using
cloned DNA and primers of defined sequence.
❑ Usually the reaction conditions must be varied to improve the
efficiency.
❑ If the reaction is not optimal, PCR often generates a smear of
products on a gel rather than a defined band.
❑ The usual parameters to vary include the annealing temperature
and the Mg2+ concentration.
❑ Too low an annealing temperature favors mispairing.
❑ The optimal Mg2+ concentration varies with each new sequence, but
is usually between 1 and 4 mM.

LO 12.6: Students are able to explain PCR optimization

 PCR variations

❑ Variations on basic PCR include

❑ Nested PCR
❑ Multiplex PCR
❑ Inverse PCR
❑ Rapid Amplification of cDNA Ends (RACE)
❑ PCR mutagenesis
❑ Reverse transcriptase (RT)-PCR
❑ Quantitative PCR
❑ Real time PCR.

LO 12.7: Students are able to explain PCR variations

 Nested PCR
❑ The specificity of the reaction can be improved by carrying
out nested PCR, where, in a second round of PCR, a new
set of primers are used that anneal within the fragment
amplified by the first pair, giving a shorter PCR product.
❑ If on the first round of PCR some nonspecific products have
been produced, giving a smear or a number of bands, using
nested PCR should ensure that only the desired product is
amplified from this mixture as it should be the only sequence
present containing both sets of primer-binding sites.

LO 12.7: Students are able to explain PCR variations

 Multiplex PCR
❑ If multiple pairs of primers are added, PCR can be used to
amplify more than one DNA fragment in the same reaction
and these fragments can easily be distinguished on gels if
they are of different lengths.
❑ This use of multiple sets of primers is called multiplex PCR
and is often used as a quick test to detect the presence of
microorganisms that may be contaminating food or water, or
be infecting tissue.

LO 12.7: Students are able to explain PCR variations

 Inverse PCR
❑ Modifications to the basic PCR make it possible to amplify
(and hence clone) sequences that are upstream or
downstream of the region amplified by the basic primer pair.
❑ For example, if genomic DNA is first digested by a restriction
enzyme and then circularized by ligation, a pair of
back-to-back primers can be used to amplify round the circle
from the region of known sequence to obtain the 5′- and
3′-flanking regions up to the joined restriction sites.
❑ This is known as inverse PCR.

LO 12.7: Students are able to explain PCR variations

 Rapid amplification of cDNA ends (RACE).
❑ When a fragment of cDNA has been produced by RT-PCR it
is possible to amplify the 5′-flanking sequence by first using
terminal transferase to add a tail, e.g. oligo(dC), to the first
strand cDNA.
❑ This allows a gene specific primer to be combined with
oligo(dG) primer to amplify the 5′-region.
❑ This technique is called rapid amplification of cDNA ends
(RACE).
❑ 3′-RACE to amplify the 3′-flanking sequence of eukaryotic
mRNAs uses a gene specific primer and an oligo(dT) primer
which will anneal to the poly(A) tail at the 3′-end of the mRNA.

LO 12.7: Students are able to explain PCR variations

 PCR Mutagenesis
❑ PCR can be used to
❑ make labeled probes to screen libraries or carry
out blotting experiments, by adding radioactive or
modified nucleotides in the later stages of the
PCR reaction, or labeling the PCR product
generated.
❑ PCR can also be used to introduce specific
mutations into a given DNA fragment.
❑ This is known as PCR mutagenesis.

LO 12.7: Students are able to explain PCR variations

 Reverse transcriptase (RT)-PCR
❑ This method of reverse transcribing mRNA and then
PCR amplifying the first strand cDNA.

LO 12.7: Students are able to explain PCR variations

 Quantitative PCR
❑ Quantitative PCR can determine the amount (number of molecules) of
DNA in a test sample.
❑ One of the best methods of quantitative PCR involves adding known
amounts of a similar DNA fragment, such as one containing a short
deletion, to the test sample before amplification.
❑ The ratio of the two products produced depends on the amount of the
deleted fragment added and allows the quantity of the target molecule in
the test sample to be calculated.
❑ In asymmetric PCR only one strand is amplified (in a linear fashion) and
when applied to DNA sequencing, it is known as cycle sequencing.
❑ PCR can also be used to increase the sensitivity of DNA fingerprinting.

LO 12.7: Students are able to explain PCR variations

 Real time PCR
❑ In real time PCR the thermal cycler can determine the amount
of product that has been made as the reaction proceeds, for
example by detecting the increase in dye binding by the
synthesized DNA, using a fluorometer.
❑ The advantages of real time PCR, apart from immediate
information on the progress of the reaction, include high
sensitivity, ability to cover a large range of starting sample
concentrations, easy compensation for different efficiencies of
sample amplification and the ease of processing many
samples, since these do not necessarily need to be analysed
at the end by, for example, gel analysis.
❑ Unfortunately the equipment is rather costly.

LO 12.7: Students are able to explain PCR variations

 PCR
▪ There was a time when to
Song amplify DNA,
▪ You had to grow tons and tons
of tiny cells.
▪ Then along came a guy named
Dr. Kary Mullis,
▪ Said you can amplify in vitro just
as well
▪ Just mix your template with a
buffer and some primers,
▪ Nucleotides and polymerases,
too.
▪ Denaturing, annealing, and
extending.
▪ Well it’s amazing what heating and cooling and heating will do.
▪ PCR, when you need to detect mutations.
▪ PCR, when you need to recombine.
▪ PCR, when you need to find out who the daddy is.
▪ PCR, when you need to solve a crime.
(repeat chorus)
https://www.youtube.com/watch?v=mvvP90Cpdfc
 Next...

LECT-13
DNA SEQUENCING