An Overview of the PCR

Yasmin Badshah
Assistant Professor
Healthcare Biotechnology
NUST
“The polymerase chain reaction (or
PCR) is a technique for the in vitro
amplification of a desired
sequence of DNA”
• PCR allows the generation of a large
quantity of DNA product from only a few
starting copies. It has been shown that
PCR can be used to generate a detectable
quantity of DNA from only one starting
target (or template) molecule.
 PCR was developed in the mid-
1980's, but has already found
multiple applications, such as:
1.Rapid amplification of intact genes or gene fragments
2.Generation of large amounts of DNA for sequencing
3.Analysis of mutations for medical applications
4.Amplification of chromosomal regions adjacent to
genes of known sequence
And many more·
Development of PCR won the Nobel prize for Kary Mullis
and co-workers.
PCR….
1. PCR allows the specific synthesis of a
predetermined DNA region via the use of two
small, specifically designed fragments of DNA
(primers or oligonucleotides),
2. two termini nucleic acid molecule amplified.
3. PCR amplification reactions in general are highly
specific, specificity being determined by the
correct hybridisation of primer specific sequences
to complementary sequences present on the
target DNA molecule to be amplified
steps in PCR
• There are three major steps in a PCR,
which are repeated for 30 or 40 cycles.
This is done on an automated cycler, which
can heat and cool the tubes with the
reaction mixture in a very short time.
1. Denaturation at 94°C :
all enzymatic reactions stop (for example :
the extension from a previous cycle).
2. Annealing at 54°C :
Brownian motion.
• Ionic bonds are constantly formed and
broken
• The more stable bonds …..on that little
piece of double stranded DNA …. the
polymerase can attach….starts copying the
template.
3. extension at 72°C :
ideal working temperature for the
polymerase.
• The primers……already have a stronger
ionic attraction to the template than the
forces breaking these attractions.
• Primers that are on positions with no exact
match, get loose again
• The bases (complementary to the template)
are coupled to the primer on the 3' side
 PCR theory
“The PCR reaction is a DNA synthesis
reaction that depends on the extension of
primers annealed to opposite strands of a
dsDNA template that has been denatured
(melted apart) at temperatures near
boiling. By repeating the melting,
annealing and extension steps, several
copies of the original template DNA can be
generated”
Brief Overview of PCR Requirements
 PCR Reaction Mix
1) Target DNA
2) specifically primers
3) Deoxyribonucleotide triphosphates
4) magnesium ions
5) Thermostable DNA polymerase (Thermus
thermophilus, T. flavus, T. litoralis, T.
brokianus)
6) water.
7) RT (Avian Myeloblastoma Virus (AMV) and
Moloney Murine Leukemia Virus (MMLV)

All prepared in a total typical PCR reaction volume

of 20-50 μl
 PCR Con…..
1. composition of the PCR reaction mix may vary
dependant on the DNA polymerase used
2. Low concentrations of detergents such as Triton X-
100, Tween-20, betain or dimethylsulphoxide
(DMSO) may also be included in the PCR mix to
help increase the specificity of primer binding.
3. DMSO may assist in overcoming problems caused
by secondary structure or possibly even inhibitory
compounds.
4. The composition of the PCR mix should ideally be
optimized
5. All PCR reaction ingredients should be stored in a
freezer in a dedicated “clean” room
6. multiple PCRs in a single “batch”
 Miscellaneous Considerations

1. Several additional factors ….Success

or failure of PCR
2. Nature and use of the PCR reaction
tube
3. Changes in the quality of ingredients
affect the sensitivity and specificity of
the PCR reaction
4. Controls are particularly important
DNA Extraction /RNA Extraction
The Different Types and Varieties
of Nucleic Acid Target Molecules
• must be readily accessible to primers and
DNA polymerase and free from inhibitory
concentrations of contaminating proteins,
lipids, carbohydrates and salts
• Blood samples
• RNA
• DNA
• Serum samples
 A Brief Description of DNA and
RNA Targets
A.Bacteria
B.Viruses
1.Class I: Double stranded DNA viruses. human
papilloma viruses (cervical cancer). Replicate in the
host cell cytoplasm.
2.Class II: Single stranded DNA viruses One example is
Parvovirus B19
3.Class III: Double stranded RNA viruses example is
rotaviruses
4.Class IV: single stranded RNA viruses example is
Poliomyelitis virus
5.Class V: single stranded RNA viruses, Influenza
viruses
6.Class VI: Retroviruses, HIV
 Contaminations….
• Blood
• Inhibitors
• kits
• RNase
1.gloves should be worn
2.use of disposable RNase free plastic…….
3.work surfaces are also …RNase free
4.inactivated by….. DEPC (diethyl pyrocarbonate) or
commercially available …… RNase AWAY, RNase ERASE,
RNA Clean
5.DEPC in water being used to remove RNase
6.DEPC also hydrolyses the imidazole ring of purines and
destroys nucleic acids.
7.180°C to inactivate the DEPC
8.DEPC….toxic carcinogen that……….safety
precautions
• DNase
Primers
 Primers
• Hybridisation
• provide the 3′ hydroxyl ends
• position of the primers along DNA strands
defines length of the PCR amplification product
generated Primers
• PCR amplification products (100–200
nucleotides), long PCR amplification products
(3,000–5,000 bp)
• primer annealing sites on the target DNA must
be known
• 18–22 nucleotides
 Primers…..
1. hybridise to either the parallel or anti-
parallel strand
2. need to be precisely complementary to
their target sequences, some sequence
data from the terminal ends of the DNA
is required for primer design (Fig. 1.1).
3. Once hybridised …. 3¢-hydroxyl
terminus required by DNA polymerases
4. effectively acting as Okazaki fragments
 PCR Primer Design and Quality
Requirements
1. base composition
2. length of primer
3. GC-content
4. annealing temperatures,
5. no internal complementary regions
6. Complementary regions…..primer/primer
hybridisation…… “primer dimer”
7. secondary structure….. primer dimers
8. primer-dimer formation…..multiplex PCR
protocols,
9. Y=G, z=C, x=T, and w=A
 main rules for effective
primer design
• 18 and 30 nucleotides (bp) in length.
• no internal complementary regions (e.g.
multiple guanosine and cytosine repeats)
• GC content 50%
• genetically stable….. Conserved sequences
• inosine nucleotide
• annealing temperatures…65°C
• annealing temperatures ….. ±5°C
 Primer Hybridisation Depend
(Annealing)
• annealing time….dependent on the ramp rate
• heating capacity…..PCR thermocycler used
• volume of the PCR mix
• the concentrations of the primer and target
template
• composition of the sample buffer
• primer length
• GC content
• reaction tubes
• Tm = 2 (T+A) + 4 (G+C)
 Primer Synthesis

• Commercial companies
• nucleic acid synthesising machines (e.g.
the ABI 3400 DNA synthesizer, USA).
Effect of Mismatches Between PCR
Primer and Target
1. Mutations in the target sequence or incorrectly
designed PCR primers
2. resulting in incomplete base pairing between the
two nucleic acid molecules…..
3. lowers the annealing temperature
(Tm)……reducing the yield of specific PCR
amplification products
4. base pair mismatches 3¢-end of the primer ……
larger impact than those near the 5¢-end of the
primer
 Primer Concentration
• 0.1–1 μM concentration ….. (in 50 μl)
• but excess of primer…….non-specific PCR
products so
• Increasing annealing temperature
• lowering the initial primer concentration
• primer concentration versus magnesium ion
concentration
Deoxynucleotide Triphosphates
(DNTPs)
DNTPs….
1. building blocks of nucleic acid molecules
2. necessary components of PCR mixes
3. The four individual deoxynucleotides…in PCR
1. Deoxyadenosine triphosphate, dATP
2. Deoxythymidine triphosphate, dTTP
3. Deoxycytosine triphosphate, dCTP
4. Deoxyguanosin triphosphate, dGTP
4. purchased either individually or as an mix
5. stable when stored at −20°C
6. Acidic in nature…so...working and stock solutions …. to
be neutralized with alkaline compounds
7. 10 mM stock solutions
8. dNTPs may be lost due to non-specific heat
inactivation…… during a 40 cycle PCR program….. loss
of dNTPs may be in 50% of the original amount added
to the PCR mix
 DNTPs….
• dNTPs…pH 7.2...negative charge….ability to
bind both mono- and divalent cations…..mg
ions
• Mg ions …. bound by dNTPs
 Factors Affecting the Choice of
dNTP Concentration
• use a concentration of dNTPs …. only a fraction
at the end of the PCR cycling
• if the dNTP limiting...rate of dNTP incorporation
will be reduced
• 10μl……..0.2 μl
 Modified dNTPs
Typical PCR protocol….all four dNTPs
1. specially modified dNTPs may also be added to
the PCR
2. helix destabilizing nucleotide>>7-deaza- 2′-
deoxyguanosine
3. 7-deazaguanine forms a normal Watson–Crick
base pair with cytosine, detailed thermodynamic
and structural analyses of this modification have
not been.
4. helps to overcome amplification problems
5. solve problems related to sequence variation
within the target DNA.
6. useful in amplifying CpG islands (GC rich
sequence found in the promoter sequences of
many higher eukaryotes)
Disadvantage of the use of
Modified dNTPs
• 7-deaza-2′-deoxyguanosine is very difficult
to stain using ethidium bromide.
• So dGTP is also added to the PCR mix at
a concentration 25%
• their presence modifies restriction digest
recognition sites…. these sites may no
longer be recognized by restriction
enzymes.
 M-nucleoside……
• Alternatively, another universal nucleoside (1-
(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole) has
been described in the literature
• This nucleoside is known as “M-nucleoside”
• maintains the ability …. four normal dNTPs
• its incorporation in PCR primers affects the Tm
of primer annealing much less markedly than
inosine