Practicals
25-2-15
Traceability in different food products through modern analytical techniques:
PCR
Polymerase Chain Reaction (PCR) developed by Kary Mullis.
The possibility of generating great quantities of DNA by amplifying fragments of genomic or
cloned cDNA has increased the possibility of screening gene-banks, analyzing mutation,
mapping chromosomes and thousands of other applications (Saiki et al., 1985).
PCR, the repetitive bi-directional DNA synthesis via primer extension of a region of nucleic
acid, is simple in design and can be applied in endless ways.
PCR amplification of a template requires two oligonucleotides primers, the four
deoxynucleotides triphosphates (dNTPs), Magnesium ions in molar excess of the dNTPs, and a
thermostable DNA polymerase to perform the DNA synthesis (Dieffenbach and Dveksler, 1995).
PCR reaction has a great efficacy, but this must be measured also by its specificity, efficiency
and accuracy that depend on a number of parameters.
The initial PCR procedure described by Saiki et al. (1985) used the Klenow fragment of
Escherichia coli DNA polymerase I. This enzyme was heat labile and fresh enzyme had to be
added.
Introduction of the thermo-stable Taq polymerase, the DNA polymerase obtained from Thermus
aquaticus, in PCR (Saiki et al., 1988) resolved this problem and made possible the automation
of the thermal cycling in the procedure.
Virtually all forms of DNA and RNA are suitable substrates for PCR. These include genomic,
plasmid, and phage DNA, previously amplified DNA, cDNA, and mRNA.
Samples prepared via standard molecular methodologies (Sambrook et al., 1989) are sufficiently
pure for PCR, and usually no extra purification steps are required.
In general the efficiency of PCR is greater for smaller-size template DNA than for high
molecular weight DNA.
How PCR techniques work
The PCR reaction allows the million-fold amplification of a specific target DNA fragment.
The PCR is a multiple-process with consecutive cycles of three different temperatures.
In each cycle (Fig. 1) the three temperatures correspond to three different steps in the reaction
(Dieffenbach and Dveksler, 1995).
In the first step the template, the DNA serving as master copy for the DNA polymerase is
separated into single strands by heat denaturation at ~95oC.
In the second step the reaction mix is cooled down to a temperature of 50±60 oC (depending on
the composition of the primers used) to allow the annealing of the primers to the target sequence.
In the third step, the annealed primers are extended using a Thermus aquaticus (Taq) polymerase
at the optimum temperature of 72 oC. With the elongation of the primers, a copy of the target
sequence is generated.
The cycle of these three different temperatures is then repeated from 20 to 50 times, depending
on the amount of DNA present and the length of the amplicon (amplified DNA fragment).
Fig. Three steps in PCR amplification: Temperatures in the hybridisation (or annealing) step may
vary.
PCR-based detection depend on the selection of the oligonucleotide primers and the knowledge
of the molecular structure and DNA sequences used.
Faster cycling with better temperature control using capillaries in air heated thermal cyclers has
improved PCR specificity.
`Rapid cycle' PCR requires amplification cycles of 20±60 sec and the whole procedure of
amplification, 30 cycles, only 10±30 min.
Rapid cycle is based on a `kinetic' rather than an `equilibrium' paradigm for PCR.
In equilibrium mode 3 reactions occur at 3 temperatures for 3 times during each cycle, in the
kinetic mode both temp transition and denaturation and annealing are in a constant state of
change.
Confirmation/ verification of the identity of the amplicon is necessary to ensure that the
amplified DNA really corresponds to the chosen target sequence and is not a by-product of unspecific binding of the primers.
For this purpose several methods are available such as gel electrophoresis to verify if the PCR
products have the expected size and purity.
Analyzing PCR products during amplification is known as `real-time PCR'.
Another possible control is to subject the PCR product to a second round of PCR. This technique
is called nested PCR. This technique reduces nearly to zero the possibilities of un-specific
amplifications (Zimmermann et al., 1994).
The most reliable way to confirm the authenticity of a PCR product is its sequencing. This
method depends on DNA quality and purity.
DNA isolated from processed foods and certain agricultural matrixes is usually of low quality
and available target sequences may be rather short. DNA purity can also be severely affected by
contaminants in food matrices (Ahmed, 2002).
Taq Polymerase, the key enzyme used in the PCR reaction is inhibited by contaminants such as
polysaccharides, EDTA, phenol and SDS. All these compounds can thus affect the amplification
reaction.
Qualitative and quantitative PCR techniques
Qualitative techniques
PCR has been used in many different applications because it has a very great flexibility in the
field of molecular biology.
Its principal use is to generate a large amount of a desired DNA product starting from a given
template, but it can be used also to amplify very long fragments of DNA and in such a way to
synthesize whole genes, to amplify and quantify specific RNA species, to produce RNA
fingerprinting, or PCR mediate cloning, to screen DNA libraries and to produce DNA sequences.
Long-distance PCR
This method amplifies and detects routinely and specifically PCR products ranging in size from
less than 1Kb to more than 50 Kb (Foord and Rose, 1995), regardless of target template sequence
or structure.
The ability to amplify fragments up to 20±50Kb enables the isolation of an entire gene from a
cDNA probe.
Large genomic fragments can be isolated from complex genomes, as well as from hybrid cell
lines or from micro-dissected or flow-sorted chromosomal regions.
In situ PCR
Direct cellular localization of a DNA or RNA target was routinely achieved by in situ
hybridization.
This method has been dramatically improved in its sensitivity.
PCR starting from RNA
RNA-PCR is particularly useful if very low quantities of mRNAs are available. RNA-PCR is a
good method for screening cells and tissues for the expression of an mRNA.
RNA fingerprinting using arbitrarily primed PCR
The extension of arbitrarily primed PCR (AP-PCR) fingerprinting to RNA has resulted in a tool
with exciting potential for detecting differential gene expression (Liang and Pardee, 1992).
It is now possible to obtain a partially abundance-normalized sample of cDNA produced in a
single tube in a few hours.
PCR-mediated cloning
Under standard PCR conditions, sufficient sequence information from a template is required to
design two primers that hybridize to each strand of the DNA.
To clone a previously uncharacterized cDNA or gene fragment, a limited quantity of genetic
sequence may be available, and thus only one primer can be designed.
Under these circumstances PCR can be used to create the second site for primer annealing,
making possible to clone the desired fragment (Dieffenbach and Dveksler, 1995).
A PCR-based method for screening DNA libraries
Screening DNA libraries of high complexity for rare sequences is one of the fundamental
techniques of molecular biology.
A pool that contains the desired clone is subdivided into smaller pools, each of which is then
screened by using the PCR protocol that was applied for the first screen.
After several cycles of subdividing and screening, the initially rare clone is greatly enriched and
can be easily obtained as a pure clone.
PCR Cycle sequencing
This technique employs a thermostable DNA polymerase in a temperature cycling format to
perform multiple rounds of dideoxynucleotide sequencing on the template (Murray, 1989).
Quantitative techniques
A PCR reaction profile is characterized by three segments: an early background phase, the
exponential growth or log phase, and a plateau. During log phase the amplification proceeds
according to equation
Tn = T0 (E)n
where Tn is the amount of target sequence at the cycle n, T0 is the amount of target at time zero,
and E is the efficacy of amplification.
A major drawback of conventional PCR is the lack of accurate quantitative information due to
the effect of the amplification efficiency (E).
If the reaction efficiency remain constant, the concentration of DNA following PCR would be
directly proportional to the amount of initial DNA target.
Unfortunately, E is not a constant parameter, but varies between different reactions, particularly
in the later cycles of PCR.
Conventional PCR relies on end point measurements, when often the reaction has gone beyond
the exponential phase because of limiting reagents (Cha and Thilly, 1995).
The two principal techniques of quantitative PCR in use at the moment are: QC-PCR
(quantitative-competitive PCR) and real time PCR.
QC-PCR is the co-amplification of a target analyte with an internal standard. It involves the coamplification of unknown amounts of an internal control template in the same reaction tube by
the identical primer pair (Studer et al., 1998).
Multiple PCR reactions are needed as each sample is amplified with increasing amounts of
competitor, while maintaining constant the sample volume/concentration.
Quantification is achieved by comparing the equivalence point at which the amplicon from the
competitor gives the same signal intensity of the target DNA on stained agarose gels (Studer et
al., 1998; Hardegger et al., 1999).
Real time PCR has rapidly gained popularity due to the introduction of several real-time
complete instruments and easy-to-use PCR assays.
With this technique the amplification of the target DNA sequence can be followed during the
whole reaction by the indirect monitoring of the product formation.
Real time detection strategies rely on continuous measurements of the increment in fluorescence
generated during the PCR reaction.
SYBR Green
This is a dsDNA binding dye. The dye has been employed in place of ethidium bromide as a
double stranded DNA dye to reduce background and allow better real time monitoring of
product formation.
It is thought to bind the minor groove of dsDNA and upon binding increases in fluorescence over
100-fold. It is important to note that at very high concentrations it starts to inhibit the PCR
reaction.
The disadvantage is that specific product, non-specific products and primer dimmers are detected
with SYBR Green (Wittwer et al., 1997).
Hybridisation probes
If sequence specific recognition is required, hybridisation probes allow detection only of the
specific product. In this case two probes are designed that hybridise side by side on the PCR
product.
Rules for probe design
The LightCycler Hybridisation probe method uses two fluorescent labeled oligonucleotide
probes that hybridise in a head to tail arrangement to adjacent sequences on the target DNA.
Molecular beacons
These have been successfully employed in real time PCR and for the generation of melting
curves, including the multiplex PCR format, and they are widely used for discriminating single
base pair differences (SNP).
Presently, real-time quantification can be considered as the more powerful tool for the detection
and quantification of GMOs in a wide variety of agricultural and food products (Hubner et al.,
2001).
Method validation
Analytical methods used by enforcement laboratories, especially where legal proof may
become necessary, should be subject to validation procedures for reliable and repeatable results.
The objective of validation is to demonstrate that the defined system produces acceptably
accurate, precise and reproducible results (Hubner et al., 2001).
These studies must be carried out according to harmonized international protocols.
The validation parameters for a qualitative method are:
Specificity: the probability to obtain a negative result given that the analyte is not present.
Sensitivity: the probability to obtain a positive result given that the analyte is present.
Two additional controls should be used routinely: a negative control as a test of contamination
and a positive control close to the detection limit as a test of sensitivity.
Limit of detection:
Precision: inter-laboratory variation.
Robustness: the reliability of the method should be demonstrated with respect to deliberate
variations in method parameters.
Overall Accuracy: defined as the probability to obtain a correct result.