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3.3.2
Dna-Based Testing Methods
A GMO is usually defined as a living organism whose genetic composition has
been altered by means of gene technology. This involves DNA isolation, defined DNA
modification, and transfer of DNA into the genome of the target organism that
successively becomes a GMO. This process is referred to as the transformation event.
Normally, new gene functions are inserted into the GMO, but new techniques have been
developed that make targeted knock-out of existing genes possible, among others in
higher organisms, such as food plants. A typical insert (gene construct) in a GMO is
composed of at least three elements:
(1)
the promoter element, which functions as an on/off switch for
reading of the inserted/altered gene;
(2)
the gene that has been inserted/altered, which is coding for a specific
selected feature;
(3)
the terminator element, which functions as a stop signal for reading
of the inserted/altered gene.
In addition, several other elements can be present in a gene construct and their
function is usually to control and stabilise the function of the gene, demonstrate the
presence of the construct in the GMO, or facilitate combination of the various elements
of the construct. A gene construct must be integrated in the genome (the natural genetic
background) of the organism to become stably inherited.
The integration process itself is complex and largely beyond human control and
in addition to one or more copies of the construct, fragments of the constructs may
become inserted and stably integrated into the recipient genome, depending on the
transformation strategy.
Other breeding techniques than gene technology are used to modify DNA and
increase genetic variability, such as chemical and irradiation mutagenesis. Although
these techniques also involve genetic modifications, they do not involve the set of
techniques defined as gene technology. Therefore, in most countries (not Canada and
US), such techniques were defined to be outside the regulatory framework for
authorisation and labelling for GMOs emerging in the 1990s. However, there are
several scientific issues in common for GMOs and varieties established by other
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breeding techniques. Common for all these technologies is that the genetic material is
modified with the objective of creating new and more useful varieties. A major
difference is the degree of targeting. Gene technology is considered a highly targeted
technology, which may involve few to moderate numbers of changes and mainly tend to
seek modification at a single locus, whereas alternative mutagenic technologies usually
create a large number of more or less random changes. On the other hand, a unique
feature of gene technology is that it allows for transfer of genetic material across species
barriers. The degree of targetedness is of central relevance for the detectability, since
better knowledge of the genetic changes means that it is easy to develop methods to
detect the genetic modifications.
DNA detection methods for GM foods rely on the complementarity of two
strands of DNA double helix that hybridize in a sequence-specific manner. The DNA
that has been engineered into a crop consists of several elements that govern its
functioning. They are typically a promoter sequence, structural gene and a stop
sequence for the gene. The availability of sufficient quantities of pure and intact DNA is
always a crucial point in DNA-based methods, i.e. for polymerase chain reaction (PCR),
DNA sequencing, Southern blotting, and microarrays. The same is true for the DNAbased methods for detection of genetically modified (GM) food. According to the
European legislation, food products must be labeled as genetically modified, if GM
compounds are present in proportion higher than 0.9%. GM food means food
containing, consisting of or produced from genetically modified organisms (GMO). The
majority of the existing methods for the detection of GM food is based on testing for the
presence of recombinant DNA, or on the detection of novel expressed proteins. Nearinfrared (NIR) spectroscopy is also applicable, if there are enough differences between
the conventional and the engineered food. DNA-based methods include Southern
blotting, conventional qualitative PCR, quantitative competitive PCR, and qualitative or
quantitative real-time PCR.
During the production chain, food passes several physical, biological, and
chemical processes, which mostly negatively influence the quantity and quality of
available DNA. Three different approaches for DNA isolation from plant material and
plant-derived products are the most widely used for GM food detection: the
cetyltrimethylammonium bromide (CTAB) method, DNA-binding silica columns in
form of various commercial kits and a combination of both. The existing methods for
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DNA isolation from food cannot always fulfill the expectations regarding quantity and
quality of isolated DNA. Furthermore, they usually require only up to 100 mg of food
sample and are difficult to scale-up.
Chromatographic methods were traditionally used for the isolation of proteins
from various sources, such as microbial culture, animal tissue and plants. More recently,
with emerging techniques in biotechnology and bioengineering, the isolation of
recombinant proteins, peptides, carbohydrates and nucleic acids has gained importance.
The supports for stationary phases applied in chromatography of large biomolecules
must fulfill some distinct criteria, different from those for the chromatography of lowmolecular-mass biologically non-active molecules. The major problem is the restricted
access of the biomolecules to the small pores of the classical porous chromatographic
supports. The so-called wide-pore silica columns opened a broad field of new
applications in the chromatography of biomolecules. Some problems such as the slow
mass transport resulting in peak broadening and low recovery still remained unsolved.
Another approach, which allowed overcoming the problem of mass transport, is the
application of micropellicular supports characterized by a spherical fluid-impervious
core of support material, covered by a thin retentive layer of stationary phase. Because
of the lack of pores, the surface area is low, but rapidly and efficiently available for the
mobile phase and the sample molecules. In the late 1980s, a novel type of supports has
been introduced, called monoliths. Monoliths are constituted of a single piece of
continuous and highly porous material, which is arranged in a way to form a network of
highly interconnected channels. In such media, the mobile phase is forced to flow
trough the pores. As a consequence, the mass transport is enhanced by convection, what
contribute to minimize the void volume and the peak broadening.
Five major chromatographic modes are used for the separation of nucleic acids: sizeexclusion,
anion-exchange,
mixed-mode,
ion-pair
reversed-phase
and
slalom
chromatography. Among theme anion-exchange chromatography combined with
micropellicular supports, is described as the most prominent technique so far. At neutral
pH, the hydrophobic organic bases of the double-stranded DNA molecule are inside,
whereas the two sugar-phosphate chains spiral down the outside of the double-helix
structure. The sugar-phosphate chains create a poly-anionic, highly hydrated, and
hydrophilic surface to the solvent allowing the chromatographic separation in anionexchange mode. CIM (Convective Interaction Media; BIA Separations, Ljubljana,
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Slovenia) monolithic columns allow fast and flow-unaffected separation of several
biomolecules, including nucleic acids. Anion-exchange chromatography with CIM
disks allows the separation of oligonucleotides of different size and also the isolation of
plasmid DNA from a complex matrix, i.e. E. coli cell lysate. Bacterial genomic DNA of
size up to 200 kbp can be also separated on CIM monoliths.
The specificity of currently available DNA based methods can be divided into
four categories:
1)
screening methods that can detect a wide range of GMO without
identifying the GMO,
2)
screening methods that can detect a certain type of genetic
modifications,
3)
construct specific methods that sometimes can be used to identify the
GMO,
4)
transformation event specific methods that can be used to identify the
GMO with special exceptions mentioned below.
In addition PCR-based GMO analyses usually include testing for presence of
DNA from the particular species of interest, e.g. soybean DNA. Sometimes (in the
absence of amplifiable DNA from the particular plant species) GMO analyses also
include testing for presence of amplifiable (multicopy) DNA from plants or Eukaryotes,
e.g. chloroplast DNA or nuclear ribosomal small subunit genes (18S like).
The promoter and terminator elements used to transform most of the currently approved
genetically modified plants are the Cauliflower Mosaic Virus promoter (P-35S) and the
Agrobacterium tumefaciens nopaline synthase terminator (T-Nos). Although, other
promoters and terminators have also been used, almost all GM plants contain at least
one copy of the P-35S, T-35S and/or the T-Nos as a part of the gene construct integrated
in its genome. Consequently, methods detecting one of these elements are popular for
screening purposes (category 1). One problem with these methods is that the elements
they detect are from naturally occurring virus and bacteria which are often present in
fresh vegetables or the environment in which they are grown. Such elements therefore
pose a significant risk of yielding false positive results.
The various genes inserted in a GMO may characterize a group of GMO, although they
may not identify the GMO. Detection of the synthetic specific gene coding for the
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Bacillus thuringiensis endotoksin CryIA(b) demonstrates the presence of a genetically
modified maize, but the gene has been used in more than one GMO. Consequently,
category 2 methods like those that can detect the CryIA(b) gene can tell us more than
the category 1 screening methods, but will not be suitable for identification of the
specific GMO.
The synthetic CryIA(b) gene has been integrated with different specific regulatory
elements (promoters and terminators) in the various GMO containing the gene.
Currently it is therefore possible to identify the GMO with category 3 detection methods
targeting the junctions where the gene and regulatory elements are fused. However, in
the future, even these junctions may be found in more than one GMO.
With currently available technology it has not yet been possible to control where in the
genome of a plant the insert is integrated. If the same insert is integrated into the
genome of the same type of organism several times, the likelihood of integration of the
insert in the same place in the genome two times is usually negligible. Consequently,
the junction between the integration site and the insert will be unique for each
transformation event. Category 4 methods detect these regions and will remain specific
for the transformation even when the same construct has been integrated into the same
plant species many times. However, category 4 methods are under development, and
none has yet been published.
When two GMO are crossed, e.g. two different approved genetically modified maize
cultivars, the resulting hybrid offspring may possess the genetic modifications from
both parent cultivars. This phenomenon is called "gene stacking". In the U.S.A. this
type of hybrid GMO is not regulated, because both parent cultivars are approved. In the
European Union, however, the hybrid is considered to be a new GMO and requires
separate approval. None of the above four categories of analysis methods will be able
to identify cases of gene stacking. Instead, cases of gene stacking will give results
indistinguishable from the separate detection and identification of each of the parental
cultivars in the sample.
Table 1. Examples of published methods to detect GMO derivatives grouped according
to categories of specificity
Type of method
Target sequence
Reference
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Method to detect
plant derived DNA
Chloroplast tRNALeu gene (trnL) intron
Taberlet & al.,
1991
Corn/maize single copy invertase gene
Ehlers & al., 1997
Soybean single copy lectin gene
Meyer & al., 1996
Tomato single copy polygalacturonase gene
Busch & al., 1999
Cauliflower mosaic virus promoter (P-35S)
Pietsch & al., 1997
Nopaline synthase terminator (T-Nos)
Pietsch & al., 1997
bar (phosphinotricin acetyltransferase) gene
Ehlers & al., 1997
CryIA(b) gene (synthetic)
Ehlers & al., 1997
Methods to detect
specific plant
species
Screening methods
(category 1)
Gene specific
methods (category
2)
Vaïtilingom & al.,
1999
Construct specific
methods (category
3)
Bt11 maize: junction alcohol dehydrogenase
1S intron IVS6 (enhancer) - CryIA(b) gene
Matsuoka & al.,
2001
Bt176 maize: junction CDPK (calcium
dependent protein-kinase) promoter synthetic CryIA(b) gene
Hupfer & al., 1998
GA21 maize: OTP (enhancer) - epsps gene
(RoundupReady tolerance)
Matsuoka & al.,
2001
Mon810 maize: junction P-35S - heat shock
protein (hsp) 70 intron I (enhancer)
Zimmermann &
al., 1998
Mon810 maize: junction hsp 70 intron CryIA(b) gene
Matsuoka & al.,
2001
RoundupReady®: junction P-35S - Petunia
hybrida CTP (chloroplast transit peptide)
Wurz & Willmund,
1997
T25 maize: junction pat (phospinotricin
acetyltransferase) gene - T-35S
Matsuoka & al.,
2001
Zeneca tomato: junction T-Nos - truncated
tomato polygalacturonase gene
Busch & al., 1999
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Event specific
methods (category
4)
Bt11 maize: junction host plant genome integrated recombinant DNA
Zimmermann &
al., 2000
RoundupReady® soybean: junction host plant Berdal & Holstgenome - integrated recombinant DNA
Jensen, in press
Taverniers & al., in
press
Terry & Harris, in
press
3.3.2.1
Southern blot
The method involves fixing isolated sample DNA onto nitrocellulose or nylon
membranes, probing with double-stranded (ds)-labeled nucleic acid probes specific to
the GMO, and detecting hybridization radiographically, fluoremetrically or by
chemiluminescence. Earlier probes were labeled with
32
P. However, nonradioactive
fluorescein-labeled DNA, digoxigenin-, or biotin-labeled DNA probes, with sensitivity
equal to
32
P probes, were recently used, obviating the need for radioactivity in the
testing laboratory. These nonradioactive probes reduced detection to <1 h, as opposed to
24 h labeling required by
32
P. However, because only one probe is used, and no
amplification is carried out, this method is considered less sensitive than PCR, which
employs DNA of two primers.
Recently, an alternative Southern blot technology has been attempted with near
infrared (NIR) fluorescent dyes (emitting at ~700 and 800 nm) coupled to a
carbodiimide-reactive group and attached directly to DNA in a 5 min reaction. The
signals for both dyes are detected simultaneously (limit in the low zeptomolar range) by
two detectors of an infrared imager, something not yet possible with conventional
radioactive or chemiluminescent detection techniques.
3.3.2.2
Polymerase Chain Reaction
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The investigation of an organism's genome was greatly enhanced during the early
1970s with the development of recombinant DNA technology. This technology allows
for In vivo replication (amplification) of genomic DNA regions that are covalently
linked with bacterial plasmid or virus clones. In 1985-86, a second major development
occurred at Cetus Corporation, USA, where researchers developed an in vitro method
for the amplification of DNA fragments, referred to as the polymerase chain reaction
(PCR). The idea for PCR is credited to Kary Mullis, who along with five other researchers
demonstrated that oligonucleotide primers could be used specifically to amplify specific
segments of genomic DNA or cDNA. Mullis was awarded the Nobel Prize in
chemistry in 1993 for his contribution to the development of PCR. This PCR is
based on the features of semiconservative DNA replication carried out by DNA
polymerases in prokaryotic and eukaryotic cells. PCR results in the selective
amplification of a chosen region of a DNA molecule. In this technique, DNA molecule
is not cloned in a bacterial plasmid or virus. The only requirement is that the sequence
at the borders of the selected DNA region must be known so that two short
oligonucleotides can anneal to the target DNA molecule for amplification. These
oligonucleotides delimit the region that will be amplified. In normal DNA replication
within a cell, DNA duplex opens up and a small single-stranded RNA primer is
synthesized to which DNA polymerase adds further nucleotides. In PCR, a specific
region of DNA, which is flanked by two oligonucleotide (deoxy) primers that share
identity to the opposite DNA strands, is enzymatically amplified. Amplification of
selected region from a complex DNA mixture is carried out in vitro by the DNA
polymerase I from Thermus aquaticus, a bacterium that lives hot springs, This
amplification is achieved by a repetitive series of cycles involving three steps:
1. Denaturation of a template DNA duplex by heating at 94°C.
2. Annealing of oligonucleotide primers to the target sequences of separated DNA strands
at 55-65°C.
3. DNA synthesis from the 3'-OH end of each primer by DNA polymerase at 72°C.
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By using repetitive cycles, where the primer extension products of the previous
cycle serve as new templates for the following eye the number of target DNA
copies has the potential to double each cycle (Fig.2).
This technique became important with the discovery of Taq DNA
polymerase by Kary Mullis. Taq DNA polymerase is a thermostable enzyme, which
retains activity even after denaturation by heat treatment at 94°C, whereas the DNA
polymerase initially used for amplification was the Klenow fragment of E. coli DNA
polymerase I that could not withstand the denaturation step. Earlier, amplification
was achieved by transferring samples in water baths maintained at different
temperatures and adding fresh enzyme during each cycle of amplification. Presently
automated thermal cycling apparatus are available that can amplify a millionfold target DNA sequence with just 20 PCR cycles in few hours.
In theory, each amplification cycle should double the number of target
molecules, resulting in an exponential increase in the PCR product, However, even
before substrate or enzyme becomes limiting, the efficiency of exponential
amplification is less than 100% due to suboptimal DNA polymerase activity, poor
primer annealing, and incomplete denaturation of the templates. PCR efficiency can be
expressed as;
PCR product yieldn= (input target amount) x (1 + % efficiency) cycle number
This equation can be used to calculate that ~26 cycles are required to produce 1
µg of PCR product from 1 pg of a target sequence (10 6 amplification) using an
amplification efficiency value of 70%. [1 µg PCR product = (1 pg target) x (1 + 0.7)26.]
Polymerase chain reaction is useful because of automated instrumentation. A
standard PCR machine is basically a thermal cycling instrument. The minimum
requirement for a thermal cycler is that it be capable of rapidly changing reaction
tube temperature to provide the optimal conditions for each step of the cycle.
There are three basic designs, but a design represented by the MJ Research DNA
engines™, is the most common. It utilizes the heating/cooling pump principle of the
Pelletier effect,
which
is
based
on
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heat exchange occurring between two
dissimilar surfaces that are connected in series by electric current, Figure.3 shows
the temperature profile for a typical PCR reaction cycle run on a thermal cycler. The
ramp time and temperature accuracy are the most important parameters that must be
controlled by the thermal cycler. A hot start is a method that can be used to increase
primer specificity during the first round of DNA synthesis. In this procedure, the Taq
polymerase is held inactive at temperatures below the annealing optimum (<55°C) to
avoid extending mismatched primers. The figure also shows a graph depicting the amount
of PCR product produced as a function of cycle number. The PCR efficiency
equation applies only to the exponential phase, which represents the linear range of the
reaction when plotted on a log scale. The plateau effect results from enzyme
limitations due to decreased enzyme activity from repeated exposure to 95°C and from
stoichiometric limitations of active enzyme molecules, relative to DNA templates, within
the time frame of extension period.
3.3.2.2.1
Procedure
The polymerase chain reaction is used to amplify a segment of DNA that lies
between the two regions of known sequence where two oligonucleotides (deoxy) as
primers can bind the opposite strands of DNA due to the complementary nature of base
sequences. Taq DNA polymerase catalyzes the amplification reaction (Fig.2). The
template DNA is first denatured by heating at 94°C. The reaction mixture is then cooled to
a temperature that allows the primers to anneal to their target sequences. These
annealed primers are then extended (i.e. synthesis of DNA) with Taq DNA polymerase.
The cycle of denaturation, annealing, and DNA synthesis is repeated many times,
because the product of one round of amplification serves as template for the next, each
successive cycle essentially doubles the amount of the desired DNA product. The
major product of this exponential reaction is a segment of double-stranded DNA whose
ends are defined by the 5' termini of the primers and whose length is defined by the
distance between the primers. The products of first round of amplification are
heteroge- neously sized DNA molecules, whose lengths may exceed the distance
between the binding sites of the two primers. In cycle 2, the original strands and the
new strands from cycle I are separated, yielding a total of four primer sites vith
which primers anneal. The primers that are hybridized to the new strands from cycle I
are extended by polymerases as far as the ends of the template, leading to a precise
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copy of the target region. In cycle 3, double-stranded DNA molecules are produced
that are precisely identical to the target region. The original DNA strands and the
variable extended DNA strands will continuously be produced from o exponential
doubling of the original template but at a linear rate and become negligible after the
exponential increase of target fragments (Table.2). The reaction can be continued for
25-45 cycles. Under normal conditions, the amount of Taq DNA polymerase
becomes limiting after 30 cycles of amplification. If further amplification is required,
then a sample of the amplified DNA can be diluted and used as a template for
further rounds of synthesis in a fresh PCR reaction.
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Figure 2. Schematic Diagram Of The Polimerase Chain Reaction
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Figure 3. a. Temperature Profile of a Typical PCR Cycle, b. Accumulation of PCR
target molecules as a function of cycle number.
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Table 2. PCR amplification of a target fragment with increasing number of the cycles
3.3.2.2.2
Components in a polymerase chain reaction
1. Oligomer primers: The amplification product of a PCR reaction is determined by the
sequence of PCR primers. The primers anneal to complementary sequences on the DNA
template and thereby determine the boundaries of the amplified product. Usually
primers are used at a concentration of 1 µM, which is sufficient for at least 30 cycles of
amplifications. It is assumed that the Taq DNA polymerase begins to work as soon as
priming oligomers be- come bound to their templates at low temperatures (35-55°C).
Once a DNA target has been chosen, there are several rules of thumb for primer design
that are important to consider,
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(a) Primer length: Primers for the PCR should be 16-24 nucleotides long, and
are generally, referred to as primers 1 and 2 for the two ends of target sequence. Primers
of this much length maintain specificity (chance of finding the same sequence by
chance is only 1 in 420) and provide sufficient base pairing for stable duplex formation,
(b)Duplex stability: Both the primers in a PCR reaction should have similar
melting temperatures (Tm) to ensure that they will have the same hybridization kinetics
during the temperature annealing phase. Primers with an overall G + C content of 4555% are most common. The Tm of each oligonucleotide can be calculated using an
empirical formula that takes into account base composition and the effect of nearest
neighbor interactions (the Tm of AAG is different from AGA). For primers with a low
GC content, it is desirable to choose a long primer so as to avoid a low melting
temperature.
(c)Noncomplementary primer pairs: The two primers should not share
complementarity at the 3' ends because they will give rise to primer dimer products. For
example, if one primer has the sequence 5' — …GGCG — 3' and the other primer has a
terminal sequence 5' — ...CCGC — 3', then they can form a short hybrid that will
become a substrate for DNA synthesis. Once the primer dimer product is formed, it is a
competing target for amplification,
(d) No hairpin loops: Each primer must be devoid of palindromic sequences that
can give rise to stable intrastrand structures that limit primer annealing to the template
DNA.
(e) Optimal distance between primers: This rule is specific for application, but
for most diagnostic PCR assays, it is best when the opposing primers are spaced 150500 bp apart,
(f) Sequences with long runs (i.e. more than three or four) of a single nucleotide
should be avoided. Presently, a variety of computer algorithms has been developed,
which takes all these parameters into account, and the researcher only needs to provide
a DNA target sequence.
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2. Amplification buffer: The standard buffer for PCR reaction contains KCI, TrisCI, and
1.5 mM magnesium chloride. When incubated at 72°C, the pH of the reaction drops by
more than a unit. To maintain the pH, presence of divalent magnesium cations is
critical.
3. Deoxyribonucleoside triphosphates: dNTPs are used at a saturating concentration of
200
µM for each dNTR
4. Target sequence: Template DNA containing the target sequences can be added in a
single or double- stranded form. Linear target sequences are amplified better as
compared to closed circular DNAs. Purity of template is flexible and even crude DNA
extracts (e.g. pulp of a fossil tooth, a hair follicle) can be used as long as the addition of
template does not inhibit the activity of polymerase. The concentration of the target
sequence in the PCR reaction is generally in nanograms (5-100 ng).
5. Taq DNA polymerase: Two forms of Taq DNA polymerase are available: the native
enzyme purified from Thermus aquaticus and a genetically engineered form synthesized
in
E. coli (Ampli Taq-™). Taq DNA polymerase is the most frequently used and
preferred en- zyme. The original PCR protocol used the Klenow fragment of E. coli
DNA polymerase I to perform the primer extension reaction. However, this meant that
fresh enzyme had to be added after each round of denaturation because this enzyme is
easily heat inactivated. Another problem with E. coli DNA polymerase is that the
optimal activity level of the enzyme is 37°C, which greatly limits the specificity of the
reaction due to degenerate primer annealing at this low temperature. Both these
problems were solved by the use of Taq DNA polymerase. This enzyme retains activity
even after repeated exposure to a temperature of 95°C, and is also fully active at ~75°C,
which essentially eliminates degenerate heteroduplex formation. More recently, several
other thermostable DNA polymerases have been isolated and characterized that offer
advantages for specialized PCR assays (Table.3). The biggest difference between these
enzymes is their inherent 3'->5' exonuclease proof reading activity, which is important
if
the sequence of PCR product must be error free. DNA polymerases with 3'->5'
exonuclease
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activity are Tli from Thermococcus litoralis and Pfu from Pyrococcus furiosus, which
have a 10-100 times lower error rate than DNA polymerases lacking this activity. Taq
DNA polymerase and E. coli DNA polymerase I encode a 5'-»3' exonuclease
activity.Two other parameters are processivity (number of nucleotides polymerized
before template dissociation) and extension rate (nucleotides polymerized/second).
Some thermostable DNA polymerases can use RNA templates as a substrate, which can
be useful for PCR applications that require a
separate cDNA synthesis reaction using viral reverse transcriptase. An enzyme of this is
the recombinant form of Tth polymerase (rTth) from Thermus thermophilus, which can
catalyze high temperature transcription of RNA in the presence of MnCI2.
Approximately 2 units of either of the enzymes are required to catalyze a typical PCR.
Addition of excess enzyme may lead to amplification of nontarget sequences.
Table 3. Characteristic features of a different thermostable DNA polymerases
Both forms of the polymerase (Taq and Ampli Taq™) carry a 5'->3' polymerase
dependent exonuclease activity, but they lack a 3'->5' exonuclease activity. It means that
Taq poly- merase does not correct the incorporation of mismatched bases.
The reaction is carried out in a sterile 0.5-ml microfuge tube by mixing the
components in the order of sterile water, amplification buffer, dNTPs, primers 1 and 2,
template DNA and finally the Taq DNA polymerase. Total volume can be 50 or 100 µl
The reaction mixture is overlayered with 2-3 drops of mineral oil. This prevents
evaporation of the sample during repeated cycles of heating and cooling. But now PCR
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thermal cyclers are available with heated lid; thus, mineral oil is not required in those
reactions. Also, reactions can be carried out in a much smaller volume of 10-25 µl.
The amplified DNA product is detected by agarose gel electrophoresis, followed
by staining with ethidium bromide.
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