Molecular Biology: PCR techniques
Molecular Biology:
PCR Techniques
Author: Prof Estelle Venter
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
INTRODUCTION........................................................................................................................................... 2
The principle of PCR ................................................................................................................................ 2
MATERIALS AND METHODS ..................................................................................................................... 5
Components needed ................................................................................................................................ 5
The different steps in PCR ....................................................................................................................... 6
CONTAMINATION ...................................................................................................................................... 12
OTHER PCR’s ............................................................................................................................................ 13
Reverse transcription PCR .................................................................................................................... 13
Random amplification of polymorphic DNA ........................................................................................... 14
Multiplex PCR ........................................................................................................................................ 14
Nested PCR ........................................................................................................................................... 14
Touchdown PCR .................................................................................................................................... 14
Hot Start PCR ........................................................................................................................................ 15
Real-time PCR ....................................................................................................................................... 15
(DIAGNOSTIC) APPLICATION OF PCR ................................................................................................... 15
THECHNIQUES USED IN PCR DIAGNOSTICS........................................................................................ 17
FAQs ........................................................................................................................................................... 18
REFERENCES ............................................................................................................................................ 20
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Molecular Biology: PCR techniques
INTRODUCTION
The polymerase chain reaction (PCR) is a simple method for producing unlimited copies of a specific
DNA sequence in a test tube which allows a “target” DNA sequence to be selectively amplified several
million-fold in just a few hours. The PCR achieves amplification of a predetermined fragment of DNA,
(the target; which can e.g. be from 100 – 1000 bp long) with the apparent disadvantage that the
sequences flanking the target region must be known, the latter precludes the use of PCR from
analysis of DNA regions that have not previously been studied by standard methods.
The principle of PCR
The PCR is used to amplify a sequence of DNA using a pair of oligonucleotide primers each
complementary to one end of the DNA target sequence. High temperatures are used to separate the
DNA molecules into single strands, and the synthetic sequences of ss DNA (18-30 nucleotides) serve
as primers. One primer is complementary to the one DNA strand at the beginning of the target region;
a second primer is complementary to a sequence on the opposite DNA strand at the end of the target
region.
5’-TTAACGGGGCCCTTTAAA..target sequence..TTTAAACCCGGGTTT-3’
Positive DNA strand
5’-TTAACGGGGCCCTTTAAA-3’.......................>Primer 1
and:
<.........................................................................3’-AAATTTGGGCCCAAA-5’
Primer 2
3’-AATTGCCCCGGGAAATTT..target sequence..AAATTTGGGCCCAAA-5’
Negative DNA strand
Location of PCR primers
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Molecular Biology: PCR techniques
These are extended towards each other by a thermostable DNA polymerase in a reaction cycle of
three steps: denaturation, primer annealing and extension/polymerization/extension.
Figure 2. Location of primers in a PCR (Brown, 1995)
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Molecular Biology: PCR techniques
Figure 3. The three steps of the PCR
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Molecular Biology: PCR techniques
MATERIALS AND METHODS
Components needed
Template
Any source that contains one or more intact target DNA molecules can be amplified by PCR.
Many different methods of isolating and preparing the template DNA exist; however, the
extraction method chosen does depend on the source of the DNA. Sources of DNA include
e.g. blood, sperm or any other tissue, old forensic specimens, ancient biological samples or in
the laboratory, bacterial colonies or phage plaques as well as purified DNA. PCR can only be
applied if some sequence information is known so that primers can be designed (PCR
Applications Manual, Boehringer Mannheim1995).
Primers
Primers are pairs of oligonucleotides of about 18-30 nucleotides and have similar G+C contents
so that they anneal to their complementary sequences at similar temperatures.
Deoxynucleotide triphosphate (dNTP)
A generic term referring to the four deoxyribonucleotides:
dATP, dCTP, dGTP, dTTP
Enzyme
Polymerases are normally used to amplify DNA. Taq polymerase is a unique thermostable
enzyme used in the PCR. This enzyme will not denature at 95 °C and will work optimally at 72
°C.
Reaction buffer
The reaction buffer is a buffer especially prepared for the enzyme to work optimally. Most of the
reaction buffers are supplied as a 10 x stock solution. The buffer should be diluted to 1 x in the
reaction cocktail, 1:10 (v/v). Use the recommended buffer that is supplied with the specific
enzyme. Read the product information sheets that are supplied with the enzymes.
Thermocycler
A machine that can change the incubation temperature of the reaction tube automatically,
cycling between approximately 95 – 98 °C (for denaturation), 55 - 65 °C (for oligonucleotide
annealing, depending on the sequence of the primers) and 72 °C (for synthesis)
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Molecular Biology: PCR techniques
The different steps in PCR
Obtaining the template - Isolation of DNA or RNA
The first step in any PCR is to isolate the nucleic acid to be amplified, the template, from the
sample. DNA is required principally for two reasons: to enable gene banks to be made and for
analysis of the genome, most often with respect to an individual gene that is sought or has
already been isolated. RNA and in particular messenger RNA (mRNA) can also be isolated,
cDNA can be synthesized and cloned to make a cDNA library.
The primary aim of any nucleic acid isolation procedure is to inactivate endogenous nucleases
as soon as possible after the intact cell is lysed, and then to free the nucleic acid completely
from adhering protein and other macromolecules.
The isolation of DNA and RNA can be illustrated as follows:

DNA is chemically stable and not susceptible to enzymatic degradation. Isolations are
performed at room temperature. In contrast, RNA is chemically unstable and is easily
degraded by omnipresent and persistent RNases. RNA is therefore isolated as many
enzymes: as fast as possible and at low temperature. The procedures depend on the
source, but most protocols contain the following steps (Roche Molecular Biochemical’s.
PCR Applications Manual, 1999; PROMEGA: Protocols and applications guide 1996).

Cells or tissues are lysed; (1) enzymatically by the proteolytic enzyme proteinase K in the
presence of sodium dodecyl sulfate (SDS), or (2) chemically by guanidinium
isothiocyanate (GITC). Lysis of the cell, dissociation of much of the protein and rapid
denaturation of degradative enzymes can be accomplished by a single chemical, the
anionic detergent sodium dodecyl (also called lauryl) sulphate (SDS), or its close relative
sodium lauroyl sarcosinate. Sometimes e.g. for bacterial and plant cells but not for
protozoa and animal cells, degradative enzymes such as lysozyme should be added.
Nematodes and adult worms need even more harsh conditions: freezing and thawing,
hypochlorite and sonication.

Removal of proteins by extraction with phenol and or chloroform

Precipitation of DNA by ethanol and washing the precipitate to remove detergents, salts
etc.

Dissolving the DNA in TE, neutral 10 mM Tris/HCl buffer with 1 mM EDTA to bind Mg2+
and Ca2+ ions that act as cofactors of most nucleases.
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Molecular Biology: PCR techniques
Figure 4. Phenol extraction and alcohol precipitation of DNA
Phenol extraction and ethanol precipitation can often be replaced by binding DNA to glass
particles or special resins. After washing the particles with an ethanol-containing buffer, the
DNA can be eluted by TE.
For the isolation of RNA, contaminating DNA can be removed by centrifugation, acid phenol
extraction or RNase-free DNase. Many special procedures exist for the isolation of plasmid
DNA from transformed bacteria (like quick-and-dirty minipreps) and for the isolation of DNA
from agarose gel.
If DNA is to be used for PCR no extensive purification is required. Moreover, only a small
amount of DNA is sufficient to start the amplification. However, contamination with DNA from
other sources may cause misleading results. Typical quantities: 1 ml of human blood yields 20
to 50 µg DNA.
DNA embedded in agarose
DNA can be extracted from an agarose gel. This can be either restriction fragment segments
or PCR products. The segment is usually cut from the gel with a scalpel blade. One should be
very careful in doing this. Many DNA purification methods/kits exist to clean the DNA from the
gel.
The amount of template DNA added to a PCR should be:
One typically measures DNA quantity in ng, but the relevant unit is actually moles, i.e., how
many copies of the sequence that will anneal with your primers are present. Thus, the amount
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Molecular Biology: PCR techniques
of DNA in ng that you need to add is a function of its complexity
(http://irc.igd.cornell.edu/Protocols/PCR_principles.htm)

25-50 ng eukaryotic genomic DNA in a 50 µl total volume reaction

0.5 ng plasmid DNA

< 1 µl of boiled bacterial overnight culture (too much inhibits the reaction)

For re-amplification of a PCR product: 1 µl or less of the primary PCR product
If you suspect that the sample contains inhibitors of the reaction:

Dilute the sample 1:10 or 1:100

Test the inhibition by adding an aliquot of the samples to the positive-control sample
Primers
Primers are pairs of oligonucleotides of about 18-30 nucleotides and have similar G+C contents
so that they anneal to their complementary sequences at similar temperatures (Dieffenbach
and Dveksler 1995).
When designing PCR primers, the following should be taken in consideration:

Primers should be 18 – 30 nucleotides long

The target sequence should be 100 – 1000 bp (with 5000 bp as a practical limit)

There should be a balanced distribution of G/C and A/T rich domains

Primers should have 10 – 12 Gs or Cs and a Tm (melting temperature) of at least 60 °C; a
rough estimate Tm = 4 x (number of G + C) + 2 x (number of A + T). The calculated Tm
(melting temperature) for a primer pair should be balanced. Rule of thumb: Tm = 4(G+C) +
2(A+T) and –1.5 °C for every mismatch. A Tm of 55-80 °C is desired

Primers should not form secondary structures

Primers should not end with AAA –3’, and GGG-3’, etc. (with eukaryotes also avoid the
microsatellite motifs CACA and TGTG)

Primers should not form dimers. Dimers are formed by primer molecules that can
hybridize to each other because of complementary bases in their sequences. Such
primer dimers may be elongated by the Taq polymerase, even if the dimer complex is
unstable, leading to competition for PCR reagents, and potentially inhibiting amplification
of the target DNA sequence. E.g. the 3’ ends of the following hypothetical primer pair are
complementary:

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Forward primer: 5’-TGG-CTA-ATT-ATG-3’
Molecular Biology: PCR techniques

Reverse primer: 5’-GAC-TTG-ACC-CAT-3’

5’-TGG-CTA-ATT-ATG-3’ >>>>>>> extension and formation of a primer dimer

<<<<<<<< 3’-TAC-CCA-GTT-CAG-5’
The sequence of the last three nucleotides of the primer (at the 3’-end) should not be
complementary to any triplet in either primer. Check this for the 3’end of both primers. Avoid
situations as shown below, where the ATG end of the upstream primer is complementary to the
3’-TAC triplet downstream and to 5’-CAT upstream, while the 3’-GTT end of the downstream
primer is complementary to the 5’-CAA upstream:
Upstream primer
’--CAA-CAT-ATG--3’
’--CAA-CAT-ATG---------------------------------CAA-ATG------3’
’--GTT-GTA-TAC---------------------------------GTT-TAC------3’
3’--GTT-TAC------5’
Downstream primer

Primer concentration:
Primer concentrations should be between 0.1 – 0.5 µM and can be as high as 1 µM.
Higher primer concentrations may promote mispriming and accumulation of non-specific
product. Lower primer concentrations may be exhausted before the reaction is completed,
resulting in lower yields of the desired product.
Polymerase enzymes
Thermostable DNA polymerases e.g. Taq polymerase have been isolated and cloned from a
number of thermophillic bacteria and are used in PCR as they survive the hot denaturation
step. Polymerase enzymes read the DNA template and synthesize DNA.
For most applications Taq polymerase is the enzyme of choice. The “Stoffel” fragment of
AmpliTaq is analogous to the Klenow fragment of E. coli DNA polymerase I and lacks the
intrinsic 5’  3’ exonuclease activity. It is reported to be useful for multiplex PCR (PCR with
different primer pairs) and random amplification of polymorphic DNA (RAPD). There are many
polymerases depending on their application, commercially available.
Recommended concentration is 1-2.5 Units per 100 l reaction.
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Too high enzyme concentrations result in:

Non-specific background products

Decreased specificity (Roche Molecular Biochemical’s. PCR Applications Manual, 1999)
Too low concentrations result in insufficient amounts of product.
Polymerase fidelity is influenced by multiple factors, including the tendency of a polymerase to
insert the wrong nucleotide, the presence of a proofreading 3’-5’ exonuclease which can
remove mismatches and the ease with which mismatches can be extended.
Magnesium chloride (MgCl2)
Magnesium concentration influences:

Enzyme activity/fidelity

Primer annealing

Strand dissociation temperatures

Product specificity

Formation of primer-dimer artifacts
It is therefore important to determine the ideal Mg2+ concentration for each primer pair for a
PCR. The optimal MgCl2 concentration may vary from approximately 0.5 mM to 5 mM and can
be adjusted for specific reactions.
Deoxynucleotide triphosphate (dNTP)
A generic term referring to the four deoxyribonucleotides:
dATP, dCTP, dGTP, dTTP
dNTPs should be used at equivalent concentrations. Imbalanced dNTPs mixtures will reduce
polymerase fidelity. dNTPs reduce free Mg2+, thus interfering with polymerase activity and
decreasing primer annealing. A final concentration of between 20-200 M of each results in an
optimal balance in yield, specificity and accuracy.
Thermal Cycling
Initial denaturation (95 °C – 98 °C)
Denaturation is the separation of the DNA double strand into two single strands. It is very
important to denature the DNA template completely, and so many thermal cycling programs
start with a longer initial denaturation step. If the template DNA is only partially denatured it will
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tend to “snap-back” very quickly, preventing efficient primer annealing and extension or leading
to “self priming” which can lead to false-positive results.
Step 1: Denaturation step during cycling
Denaturation at 95 °C for 20-30 seconds is usually sufficient but must be adapted for the tubes
and thermocycler being used.
Step 2: Annealing (45 °C – 65 °C)
The temperature is reduced to allow the primers to anneal. The choice of primer annealing
temperature is the most critical factor in designing a high specificity PCR. If the temperature is
too high, no annealing occurs. If the temperature is too low, non-specific annealing will increase
dramatically. The actual annealing temperature depends on the primer lengths and sequences.
After annealing, the temperature is increased to 72 °C for optimal polymerization, which uses
up dNTPs in the reaction mix and requires Mg2+.
Step 3: Primer extension (72 °C)
Time depends upon the length and the concentration of the target sequence and upon the
temperature. The rate of incorporation varies between 35-100 nucleotides/sec. A 20 second
extension is sufficient for fragments shorter than 500 bp and a 40 second extension is sufficient
for fragments up to 1.2 kb.
Final extension
After the last cycle the reaction tubes are held at 72 °C for 5-15 minutes to promote completion
of partial extension products and annealing of single-stranded complementary products.
Cycle number
Most PCRs include only 25 to 35 cycles. As the cycle number increases non-specific products
can accumulate. Actual yield is less than the theoretical maximum.
The Plateau effect
This is the point in a PCR at which running more cycles does not result in a net gain of specific
PCR amplification product. This may be due to a number of different factors, including:

depletion of reaction components, e.g. dNTPs or primers

stability of the reaction components after repeated denaturation steps, e.g. dNTPs or Taq
polymerase

inhibition by end-products, e.g. pyrophosphate or duplex DNA

competition for reaction components by nonspecific products or primer dimers
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
incomplete denaturation of PCR products at high concentration
Once the plateau is reached non-specific fragments may continue to amplify exponentially;
hence, running PCRs into a plateau may result in high background or smearing.
Analysis
Agarose gel electrophoresis
Agarose for electrophoresis is purified from the agar that is used in the preparation of bacterial
culture plates. Agarose solidifies into a solid gel when it is dissolved in an aqueous solution at
concentrations between 0.5-2% (w/v). When an electrical field is applied to an agarose gel, in
the presence of salty buffer solution, electricity will be conducted and DNA fragments (which
are negatively charged) will migrate through the gel matrix towards the positive electrode at a
rate that is dependent on size and shape of the DNA fragment.
Figure 5. An example of a gel electrophoresis system
CONTAMINATION
Due to the ability of the PCR to synthesize large amounts of DNA from a single target gene, it is
critical to avoid contamination of template DNA. It is essential that the only DNA that enters the
reaction is the template added by the investigator. Thus PCR must be performed in a DNA-free,
clean environment.
Contamination of new PCR assays with old PCR products or molecular clones must be avoided, and
sample-to-sample contamination must be prevented
Approaches to prevent contamination.

The individual parts of the PCR should be physically separated into sample preparation, prePCR, and post-PCR locations. This approach should be a central part of any contamination
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control strategy and can be scaled to suit the needs of the investigator. The physical
separation of parts of the PCR process requires some additional space, money and supplies
to equip and maintain a large infrastructure. However, good laboratory practice is still
required for the prevention of sample-to-sample contamination.

Uracil DNA-glycosylase (UNG) is an enzyme which cleaves any U’s present in a DNA strand.
To use this method of contamination control, deoxyuridine triphosphate (dUTP) is substituted
for thymidine triphosphate (dTTP) in the reaction mix, so that uracil-containing DNA (U-DNA)
is produced during the PCR. UNG is then added to all new PCRs. When this enzyme comes
across any U-containing DNA strands, the U’s are cleaved, leaving the strand with gaps.
When the reaction is heated, the DNA strands fall apart and cannot be amplified, thus
removing contaminating U-DNA from the sample. This method is effective only against
contamination with dUTP-labelled PCR products (Longo, Berninger and Hartley 1990).

The use of UV-light is effective against all types of contamination. UV-A light creates free
radicals which cause oxidative damage to DNA molecules. Direct DNA damage caused by
UV-B light results from crosslinking between adjacent cytosine and thymine bases, creating
pyrimidine dimers. This approach is limited because it cannot destroy all the PCR
contaminants; it only reduces the contamination by several logs, and is less effective if the
DNA fragment is less than 300 bp.

Single-and double-stranded DNA can be denatured with chemical adducts, such as
isopropaline. These adducts prevent the contaminating DNA from serving as a substrate in
the reaction.
OTHER PCR’S
Reverse transcription PCR
This is reverse transcription of RNA followed by PCR (RT-PCR) of the cDNA (copy DNA). Since the
polymerase enzymes used in PCR can only act on DNA templates, the RNA is first transcribed to
cDNA using a commercially available reverse transcriptase enzyme. In eukaryotes, most mature
messenger RNA (mRNA) molecules are synthesized with a poly(A) tail, which protects the mRNA
molecule from enzymatic degradation in the cytoplasm and aids in transcription termination, export of
the mRNA from the nucleus, and translation. The primer used in the RT-PCR is often oligo(dT), which
will bind to the poly(A) tail. The newly synthesized DNA template can then be amplified using PCR.
The RT step can be performed either in the same tube with the PCR (one-step RT-PCR) or in a
separate one (two-step RT-PCR)
A useful application of RT-PCR is in measuring the relative amounts of mRNA in different tissues or in
the same tissue at different times. The amount of mRNA in a cell is generally taken to be a reflection
of the activity of the parent gene, so quantification of the mRNA enables changes in gene expression
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to be monitored. The latest development in this area is the use of real-time PCR where the
amplification is monitored online and in real-time (see section 4.9).
Random amplification of polymorphic DNA
Random amplification of polymorphic DNA (RAPD) is used to generate fingerprints of genomic DNA
(viruses, bacteria, fungi, plants), and relies on the use of a short arbitrarily chosen primer (Theron,
1998). If the primers used in a PCR are too short then a mixture of amplified fragments will be
obtained. Under normal circumstances this is to be avoided but it is a useful technique in
phylogenetics, the area of research concerned with the evolutionary history and lines of descent of
species and other groups of organisms. The banding pattern seen when the products of PCR with
random primers are electrophoresed is a reflection of the overall structure of the DNA molecule used
as the template. If the starting material is total cell DNA then the banding pattern represents the
organization of the cell’s genome. Differences between the genomes of two organisms whether
members of the same or different species can therefore be measured by PCR with random primers.
Two closely related organisms would be expected to yield more similar banding patterns than two
organisms that are more distant in evolutionary terms. As with many phylogenetic techniques, the
interpretation of RAPD analysis is highly complex and as yet there is no agreement regarding the way
in which the data should be handled.
Multiplex PCR
Several DNA segments can be simultaneously amplified by using multiple pairs of primers. The
primers must be chosen so that they have similar annealing temperatures. A difference of
approximately 10 °C in the annealing temperature of the two sets of primers may lead to poor or no
amplification for one or the other target (Theron, 1998).
Nested PCR
In nested PCR, two PCRs are carried out. The first PCR is as normal, yielding a primary amplicon.
The primary amplicon is used as a template in the secondary PCR which is carried out for 15 to 30
cycles using a second pair of primers that anneal to an internal area of the first amplicon (nested
primers). This method increases the specificity and effectivity of the amplification by minimizing nonspecific annealing of the two sets of primers (Theron, 1998)
Touchdown PCR
This technique entails starting with a high annealing temperature, which is gradually lowered in the
next cycles. As soon as the temperature is low enough, amplification starts with only very specific
base pairing between the primer and the template. If the temperature is then decreased further, nonspecific binding may occur but non-specific products must compete with the already formed correct
product, resulting in optimal discrimination between specific and non-specific binding. By applying
this technique the sensitivity of the PCR is increased.
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Hot Start PCR
Hot start protocols (D’Aquila et al., 1991, Erlich et al., 1991, Mullis 1991) are designed to reduce nonspecific amplification during the initial set up stages of the PCR and can be used for PCR systems
that do not work well under standard conditions. Even brief incubations of a PCR mix at temperatures
significantly below the Tm can result in primer dimer and non-specific priming.
The aim is to prevent at least one of the critical components from participating in the reaction until the
temperature in the first cycle rises above the T m of the reactants. For example in smaller assays one
of the components common to all tubes (e.g. Taq DNA polymerase) can be initially withheld and
added only after the temperature rises above 80 °C during the first denaturing step. Alternatively, a
wax bead can be melted over the bulk of the reaction mix in each tube and allowed to solidify, and the
withheld component can be pipetted on top of the wax cap. The beads melt after the initial
denaturation step, allowing all components of the PCR to mix. Alternatively, the activity of the
polymerase can be inhibited by the binding of an antibody or by the presence of covalently bound
inhibitors. These inhibitors dissociate after a high-temperature activation step, allowing the
polymerase to function normally.
Real-time PCR
Real-time PCR uses a fluorescent signal to monitor the accumulation of PCR products in a PCR
reaction in real time. The technique reduces the time required for PCR amplification and analysis and
is suited to:

monitor amplification online and in real-time

quickly and accurately quantify results by using different chemistries:

o
SYBR Green I, a dye specific for double-stranded DNA, or
o
Sequence-specific hybridization probes
detect mutations or discriminate between homogeneous and heterogeneous genotypes.
Refer to Sub-module 3 for more information on real-time PCR
(DIAGNOSTIC) APPLICATION OF PCR

To study minute quantities of DNA. From e.g. a single sperm cell, bloodstains, hair and bones of
murder victims. A fragment of up to 5 kb can be amplified and be used for a variety of purposes.

The amplification of inserts of bacterial plasmids with primers based on the flanking vector
sequence.
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
Amplification of any DNA from which the sequence is available. Databases of DNA sequences
have been established where millions of DNA sequences are freely available.

Designing primers on the basis of homologous sequence e.g. to isolate a gene from the
chimpanzee using known human primers. Degenerate primers, obtained from the sequence of the
translated protein, can also be used. (Degenerate primers have not been explained in this
course).

Study phylogeny and evolution. Sequencing data of the bacterial 16S rRNA and of the fast
evolving genomes of mitochondria and plant chloroplasts are used in this field of study. PCR
amplicons of these genes are sequenced and with the use of specific software programs
analyzed.

The use RFLP, PCR and sequencing to determine the specific serotype of e.g. a virus. This play
an important role in the epidemiology of diseases e.g. determining the source of an outbreak.
Pathogenesis of disease.

Amplification of RNA. RNA is first converted into single-stranded cDNA with the enzyme reverse
transcriptase and then used in a PCR. A useful application of RT-PCR is measuring the relative
amounts of mRNA in different tissues or in the same tissue at different times. This is mainly done
by real-time PCR.

By amplifying and sequencing of genes or using RAPD of RFLP techniques, genomes of different
organisms can be compared - Genotyping of microorganisms.

Genetic disorders can be identified by the identification and characterization of the gene
responsible for the disease.

PCR can be used in the diagnosis of cancer by the detection of mutation/s in oncogenes or
tumor-suppressor genes.

Typing of tissue at a DNA level and comparison between individuals e.g. for bone-marrow
transplantation.

Linkage analysis of genetic markers –A marker is based on a polymorphism in a population, the
existence of two or more alleles, or genetic variants. Markers can be phenotypic (genetic
diseases), on the protein level or mutations on the DNA level (point mutations, insertions and
deletions). A genetic map of a species indicates the location of genetic markers relative to each
other. Mutations in genes are genetic markers that in many cases influence the phenotype.
RFLP with Southern blotting were the first techniques to be used to identify these markers. PCRbased techniques, like microsatellites (use of short random primers), AFLP’s (amplified fragment
polymorphism) or SNP’s (single-nucleotide polymorphisms) are currently used. These variations
of the PCR are not discussed in this course).

A combination of mutations at different positions within a gene can be analyzed using PCR.
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
Forensic identification and paternity testing. By comparing genotypes of parents and offspring or
comparing DNA from a source to a specific individual, relationships can be revealed.

Gene expression. The traditional technique to detect the expression of a given gene in a given
tissue is the Northern blot analysis of mRNA. Amplification of cDNA is a rapid and sensitive
alternative provided that:
o
The amount of mRNA can be quantified, most conveniently by co-amplification of an
internal marker or the use of quantitative real-time PCR.
o
The appropriate controls are tested to check if the signal has been amplified from
contaminating chromosomal DNA.
THECHNIQUES USED IN PCR DIAGNOSTICS
Because of its sensitivity PCR has opened possibilities not available to older techniques. One of the
following techniques can be used to discriminate between different types of microbial species:

Dot spot hybridization
PCR products are immobilized on a nylon membrane and hybridized to a specific probe. This not
only increases the sensitivity, but also verifies the identity of the PCR product.

PCR- ELISA or EIA
The PCR is carried out with a biotin label on one of the primers. This allows the immobilization of
the PCR product on microtitre plates coated with streptavidin, which binds biotin. The PCR
product is then denatured, hybridized to a labelled probe and detected by immunochemical
staining.

Reverse- blotting
Oligonucleotides are immobilized on the membrane and hybridized to the labeled PCR product as
probe. This allows testing the binding of the PCR product to many different oligonucleotides.
Oligonucleotides can be immobilized in microtitre plates or in high-density arrays on a small glass
surface (microarrays).
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FAQS
1.
What is PCR?
The PCR is a simple method for producing unlimited copies of a specific DNA sequence in
a test tube which allows a “target” DNA sequence to be selectively amplified several
million-fold in just a few hours. The PCR achieves amplification of a predetermined
fragment of DNA.
2.
Can one do a PCR without knowing the target gene/organism?
To start DNA amplification, the PCR needs specific flanking primers. In order to develop
these primers, the gene sequence of the target gene/organism must therefore be known.
This can be seen as a disadvantage of the PCR.
3.
What is the highest temperature that reverse transcriptase can tolerate?
This enzyme is not stable and will denature easily. The enzyme will be unstable from 60
°C and higher.
4.
Why do we use the DNA polymerase from Thermus aquaticus in a PCR instead of
normal DNA polymerase from E. coli?
Taq polymerase can withstand high temperature – will not denature at 95 °C.
5.
Why do different PCRs have different annealing temperatures?
Annealing temperature of primers depends on the sequence of the primers. Different
PCRs will have different primer sequences, therefore different annealing temperatures.
6.
How does one determine the correct annealing temperature for primers?
Depends on the length and the sequence of the primers, the formula: Tm= 4(G+C) +
2(A+T) can be used.
7.
Which samples are ideal to be used for PCR?
All diagnostic samples can be used for PCR
8.
Which parameter would you change first if your PCR reaction gave too many
products?
If there are too many products in a PCR reaction then it suggests that primer annealing is
non-specific. The first parameter to modify would be to increase the annealing temperature
which will increase the specificity of the primers.
9.
What would you do if the PCR reaction gave very little, if any, of the correct
product?
If no product was observed it might suggest that the annealing temperature was too high.
This can be tested by lowering the annealing temperature 3-5 degrees. However, another
possibility is that the primers are not working, in that case, new primers would need to be
designed
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Molecular Biology: PCR techniques
10.
Differentiate between a hot start PCR and a touchdown PCR.
Hot start: Protection of Taq polymerase so that PCR only starts after denaturation of DNA
and at the intended specific annealing temperature and not randomly.
Touch down: Initial annealing at a higher temperature as needed for the reaction – after a
few cycles the annealing temperature is decreased – prevent non-specific binding to occur
but has to compete with the already formed correct product - use to make reaction more
sensitive.
11.
What is the plateau effect?
This is the point in a PCR at which running more cycles does not result in a net gain of
specific PCR amplification product.
12.
What formula is used for the calculation of Tm?
Tm = 2(A+T) + 4(G+C)
13.
What are the important criteria to use when designing primers?
a) Length - 18 – 30 nucleotides long
b) Have a target sequence of 100 – 1000 bp (with 5000 bp as practical limit)
c) Primer sequence - Balanced distribution of G/C and A/T rich domains
d) Have 10 – 12 Gs or Cs and a Tm of at least 60 °C
e) Do not form secondary structure
f)
Do not end with AAA –3’, and GGG-3’, etc. (with eukaryotes also avoid the
microsatellite motifs CACA and TGTG)
g) Do not form dimers at the 3’-end. Dimers formed by the last three nucleotides may be
elongated by the Taq polymerase, even if the dimer complex is unstable:
14.
PCR experiments may be disturbed by very small contaminations. Why?
During a PCR minute quantities of DNA are amplified – any foreign DNA with a remote
primer sequence similarity will be amplified.
15.
Why is the PCR negative control handled after the other samples?
To see if any carry-over DNA occurred during the preparation of the PCR.
16.
Can single-stranded DNA serve as PCR template?
Yes single stranded DNA e.g. cDNA can be used in a PCR.
17.
What is the use of Magnesium in a PCR? Should the concentration regularly be
changed?
Magnesium concentration influences:
Enzyme activity/fidelity, primer annealing, strand dissociation temperatures, product
specificity and the formation of primer-dimer artifacts.
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Molecular Biology: PCR techniques
It is therefore important to determine the ideal Mg2+ concentration for each primer pair for a
PCR. The optimal MgCl2 concentration may vary from approximately 0.5 mM to 5 mM
18.
What is reverse transcribed (RT)-PCR?
When RNA is used as PCR template, it is first converted to cDNA and the polyA in cDNA
can act as part of the template which sequence can act as a primer (- TTT)
19.
What is a nested PCR and when is it used?
It is the re-amplification of a PCR product. Normally the second PCR (nested) uses
primers developed within the first PCR product, the amplicon. It is used to increase the
sensitivity of the PCR but can also be used to distinguish between e.g. serotypes of
organisms, vaccine and wild strains etc.
20.
What is meant by the generic term dNTPs? How do dNTPs influence a PCR?
A generic term referring to the four deoxyribonucleotides: dATP, dCTP, dGTP, dTTP
Imbalanced dNTPs mixtures will reduce polymerase fidelity. dNTPs reduce free Mg 2+, thus
interfering with polymerase activity and decreasing primer annealing. A final concentration
of between 20-200 M of each results in an optimal balance in yield, specificity and
accuracy.
REFERENCES
Multimedia
A multimedia programme on CD ROM demonstrating practical skills is available: ‘Molecular Biology
and Recombinant DNA-technology’. Developed by the Department of Veterinary Tropical Diseases.
Websites
1.
http://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/
2.
http://highveld.com/f/fpcr.html
3.
http://www.protocol-online.org/
4.
http://www.dnaftb.org/dnaftb/
5.
http://irc.igd.cornell.edu/Protocols/PCR_principles.htm
6.
http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml
7.
http://www.maxanim.com/genetics/PCR/PCR.htm
8.
http://www.sumanasinc.com/webcontent/animations/content/pcr.html
9.
http://molecular.roche.com/About/pcr/Pages/ApplicationsofPCR.aspx
10.
http://www.promega.com/country.aspx?returnurl=http%3A%2F%2Fwww.promega.com%2Fres
ources%2Fproduct-guides-and-selectors%2Fprotocols-and-applications-guide%2Fpcramplification%2F
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Molecular Biology: PCR techniques
Laboratory Manuals
1.
Molecular Cloning, A Laboratory Manual, 2nd edition. J.Sambrook, E.F. Fritsch and T. Maniatis.
Cold Spring Harbor Laboratory Press. 1989.
2.
PCR Applications Manual, Boehringer Mannheim. ©1995 by Boehringer Mannheim GmbH.
Biochemica
3.
Roche Molecular Biochemical’s. PCR Applications Manual, 2 nd edition. © Roche diagnostics,
1999 GmbH, Mannheim.
4.
PROMEGA: Protocols and applications guide. 1996
Books/Articles
1.
BROWN, T A 1986. Gene cloning. Van Nostrand Reinhold (UK) Co. Ltd.
2.
BROWN, T A 1995 3rd Edition. Gene cloning – An introduction. Chapman & Hall, London.
3.
D’AQUILA, RT, BECHTEL, L.J.,VITELER, J. A., ERON, J.J., GORCZYCZ, P. and KAPLIN,
J.C., 1991. Maximizing sensitivity and specificity of PCR by preamplification heating. Nucleic
Acids Res. 19: 3749.
4.
DIEFFENBACH, C. W. and DVEKSLER, G. S. 1995. PCR primer. A laboratory manual. CSHL
Press.
5.
EHRLICH, H.A, GELFAND, D. and SNINSKY, J.J., 1991. Recent advances in the polymerase
chain reaction. Science 252: 1643 – 1651.
6.
HYDE, J 1990. Molecular parasitology. Van Nostrand Reinhold. New York
7.
KWOK, S. and HIGUCHI, R 1989. Avoiding false positives with PCR. Nature 339: 237 – 238.
8.
LONGO, MC., BERNINGER, MS. and HARTLEY JL. 1990. Use of uracil DNA glycosylase to
control carryover contamination in polymerase chain reactions. Gene 93: 125 – 128.
9.
MAXAM, AM and GILBERT, W 1980.
Sequencing end-labelled DNA with base-specific
chemical cleavage. Methods in Enzymology, 65: 499 –552.
10.
MULLIS, K B, 1991. The polymerase chain reaction in an anemic mode: How to avoid cold
oligodeoxy-ribonuclear fusion. PCR Methods Appl. 1: 1-4.
11.
SANGER, F. NICKLEN, S and COULSON, AR 1979. DNA sequencing with chain-terminating
inhibitors. Proc. Natl Acad. Sci. USA 74, 5463 – 5467.
12.
SMITH CA and WOOD EJ 1991. Molecular biology and biotechnology. Chapman & Hall,
London.
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