3.3.2.2.4
Cloning of PCR products
There are numerous situations in which a specific DNA segment must be cloned
into a plasmid vector. Although PCR generates sufficient material for analysis, when
extensive manipulations of the sequence is needed or when large amounts of DNA are
required, it is necessary to clone the PCR product. But amplified DNA fragments do not
clone efficiently because there are no convenient restriction enzyme sites to facilitate
the design of subcloning strategy. Three basic strategies are commonly used to subclone
PCR product.
1. Restriction site addition: This is done by including the nucleotide sequence of an
appro- priate restriction enzyme into the 5' ends of the primer. A PCR primer may be
designed which, in addition to the sequence required for hybridization with the input
DNA, includes an extra sequence at its 5' end. Figure 12 illustrates the addition of a
Hindlll and an EcoRI sites to the ends of an amplified DNA fragment. Four nucleotides
are placed between the hexanucleotide restriction sites and the extreme ends of the
DNA for ensuring that the restriction sites are good substrates for the restriction
endonucleases. The extra sequence does not participate in the first hybridization step;
only the 3' portion of the primer hybridizes, but it subsequently becomes incorporated
into the amplified DNA fragment. The restriction enzyme recognition site does not
interfere with template annealing during the first PCR cycle. However, all subsequent
cycles contain PCR products that have the restriction enzyme site incorporated into the
DNA termini. By digesting the PCR products with the corresponding enzymes, the
amplified DNA fragment can be cloned by a Standard ligation reaction.
1
Figure 12. Incorporation of extra sequences at the 5’ end of a primer.
2. T/A cloning: This relies on the fact that a proportion of amplified DNA fragments are
not truly blunt ended because thermostable DNA polymerases (Taq and Tth) lack 3' to
5' exonuclease activity and have a tendency to add an extra A residue at the 3' end of
each strand. This one base overhang at each end facilitates ligation to the plasmid
vectors containing a thymidine nucleotide (TMP or TTP) overhang. Although not as
efficient as restriction site addition PCR cloning, T/A cloning is easier because PCR
products can be ligated directly to the vector without additional purification steps or
enzyme cleavage reactions.
3. Blunt-end ligation: This is required if a proofreading thermostable DNA polymerase
is used and restriction sites have not been added to the primer. The basic PCR generates
double-stranded amplified product. However, the product of the reaction does not
contain a large proportion of perfectly bluntended molecules. This can be carried out in
a combined exonuclease/repair reaction with Klenow polymerase in the presence of all
four dNTPs. Taq DNA polymerase in general remains tightly bound to the 3' ends of the
DNA, so proteinase K treatment is recommended. Excess primer is removed by
preparative gel electrophoresis as a means of purifying the amplified DNA. The DNA
2
fragment can then be blunt ligated into a suitable vector (e.g. pBluescript) linearized.
The chemically synthesized primers, which form the 5' end of each strand of the DNA
do not bear 5'-terminal phosphates. Therefore, if the linearized vector has been treated
with phosphatase to prevent simple reclosure, it is essential that 5' phosphate groups be
added to the PCR product by using a polynucleotide kinase. Blunt-ligated recombinants
can be identified by loss of β-galactosidase activity using a blue-white screening
protocol.
3.3.2.2.5
Genetic engineering by PCR
In many cases, the use of genetic regions (regulatory and coding DNAs) requires
nucleotide modifications. DNA modification techniques are numerous and include the
use of linkers, DNA modifying enzymes to change restriction enzyme sites (Klenow, T4
polymerase, etc.) and single-stranded site-specific modification methods. All these
methods are useful, but they have limitations as well as require a considerable amount
of time and effort to locate modified product. The use of PCR amplifications to modify
a DNA fragment was first discussed by Mullis for the addition of a restriction enzyme
site synthesized into the 5'-ends of PCR oligomer primers. Because this technique
requires the use of custom-synthesized oligomer primers for each DNA modification, it
is referred as custom-PCR (cPCR) engineering. The major strength of cPCR
engineering is that most of the complex DNA modifications can be incorporated into the
sequence of the synthesized oligomer primers, allowing the engineering process to
proceed as a series of straightforward ligatioin events.
3.3.2.2.6
Applications
The usefulness of PCR was quickly recognized by scientists in many different
disciplines and it, either directly or after some modifications, has been applied to
resolve many problems that were unapproachable by other techniques.
1. It has extensive applications in the diagnosis of genetic disorders such as
phenylketonuria, hemophilia, sickle cell anemia, thalassemi, etc. and the detection of
nucleic acid sequences of pathogenic organisms in clinical samples. Sometimes, the
RFLP pattern of PCR products in healthy and defective foeti can be studied or the PCR
3
product may also be sequenced to reveal the differences. For detection of pathogens,
immunochemistry and histocytochemistry have long been used by pathologists to
analyze cell phenotypes in histological specimens. However, a major limitation of these
procedures is the inability to detect infectious agents such as viral pathogens and other
microorganisms that persist at a very low level in infected cells. When it became clear
that PCR could offer increased specificity and sensitivity, researchers began to develop
PCR strategies to detect as few as one viral genome in tissue specimens. Two basic
PCR approaches have been followed for pathogen detection. The first approach is
competitive quantitative PCR, with an added emphasis on low-level detection and strain
identification. The second approach is to develop in situ PCR (ISPCR) procedures that
allow the same level of sensitivity and specificity as solution PCR assays, with the
advantage of cell-specific resolution. Similar to solution PCR, the ISPCR amplification
parameters must be optimized with regard to temperature, time, and reaction
constituents. ISPCR products are detected by using labeled dNTPs (radioactive or
biotinylated) in the reaction or by sub- sequently processing the ISPCR specimen using
standard in situ hybridization conditions to detect the amplified hybridization target.
2. The genetic identification of forensic samples, even DNA extracted from a hair or a
single
sperm are sufficient for PCR amplification. Some well-characterized sequences of
micro- satellites are being used for designing primers, so that DNA finger printing may
be achieved through PCR.
3. For the analysis of homologous genes in evolutionary biology.
4. PCR amplification has several plant molecular biology related uses. These include
i. Generation of specific sequences of cloned double-stranded DNA for use as
probes.
ii. Generation of probes for genes or cDNAs that have not yet been cloned.
4
iii. Generation of libraries of cDNA from small amounts of mRNA. The main
advantage of this method is that it allows large cDNA libraries to be established from as
few as one or two cells.
iv. Generation of large amounts of template DNA for sequencing to be carried
out either by the Maxam-Gilbert chemical degradation method or Sanger
dideoxymediated chain termination method.
v. Identification of genetic mutations: Deletions and insertions at defined loci
can be detected by a change in the size of the amplified product. Alternatively, deletions
can be recognized by their failure to amplify when one of the priming oligonucleotides
maps within the deleted sequence. It is also useful for the identification of point
mutations. The PCR technique of single-strand conformational polymorphism (SSCP) is
the widely used method for detecting single base pair changes in genomic DNA. It is
based on the principle that two single-strand DNA molecules of identical length but
unlike sequences migrate differentially in a nondenaturing acrylamide gel. This is due to
sequence-dependent intramolecular folding reactions that contribute to the overall
structure of the molecule. Single-strand conformational polymorphism is done by
amplifying a specific DNA segment using a radiolabeled (or fluorescently labeled)
primer, or by including trace amounts of
32P.-a-dNTP
in the PCR reaction. The PCR
products are then heat denatured and cooled on ice to promote rapid formation of
intrastrand duplexes. Figure.13 shows how SSCP can be used to identify mutant alleles
in known target DNA sequences. Two other PCR-based approaches for detecting single
nucleotide differences are base excision sequence scanning (BESS), which uses an
enzymatic sequencing method to detect thymidine residues in PCR products, and
artificial mismatch hybridization (AMH), a method that exploits heteroduplex stability
assays to detect single base pair mismatches.
vi. PCR detection of a gene transferred into a plant genome: The ability to
transfer genes into a plant genome is well established. The determination of those plant
tissues that are transformed from those that are not can be accomplished using
selectable marker or reporter genes in most cases. However, there are some situations in
which the use of selectable or reporter gene is not effective or where a reporter gene is
not informative, either because of its lack of expression or due to its incomplete
5
transfer. In the PCR method, genomic DNA is isolated and the PCR target gene; that is,
the gene that was used for transformation experiments is selected for amplification.
With designing of appropriate primers the gene is amplified and seen on a gel.
vii. PCR technique is employed in various molecular markers like RAPD;
microsatellites or simple sequence repeats, amplified fragment length polymorphism
(AFLP), etc. Variations observed in the amplification products are used as markers.
viii.
Identification
of
unknown
plant
DNA
regions
that
flank
an
AgrobacteriumT-DNA boundary. Although PCR is frequently used to amplify DNA
segments lying between the two priming oligonucleotides, it can also be used to amplify
unknown DNA sequences that lie outside the boundary of known sequences. This is
referred to as inverse PCR (iPCR). Inverse PCR is also useful if the border sequences of
a DNA segment are not known and those of a vector are known, then the sequence to be
amplified may be cloned in the vector and border sequences of a vector may be used as
primers in such a way that the polymerization proceeds in inverse direction, that is,
away from the vector sequence flanked by the primers and towards the DNA sequence
of inserted segment. Similarly, if the gene sequence is known, one can use its border
sequences as primers, for an iPCR to amplify the sequences flanking this gene.
6
Figure 13. Point mutation in genomic DNA can be rapidly amplified using the PCR
technique of a single strand conformational polymorpheism (SSCP).
3.3.2.2.7
Advantages
1. It can produce large amounts of identical DNA molecules from a minute amount of
starting material. In fact, the DNA isolated from a single cell (even plant protoplast) is
sufficient for gene detection.
2. The technique is quick and simple.
3. The technique is extremely sensitive.
7
4. The DNA need not be pure for amplification provided the sample does not contain
conta- minants that inhibit Taq polymerase.
3.3.2.2.8
Problems
1. Nucleotide sequence of at least the boundary regions must be known so that oligonucleotide primers can bind and synthesize the DNA. This limits PCR to the study of
genes that have already been characterized in part by cloning methods.
2. PCR is a very sensitive technique and is prone to generating false signals, which in
many cases are the result of contaminants from previous PCR amplifications being
present in the amplification reaction components. False positives can also result from
minute contamination by any plasmid or lambda clone that may contain the target gene
sequence.
3.3.2.3
NIR spectroscopy
NIR transmittance spectroscopy has been used by grain handlers in elevators in
most of the world for nondestructive analysis of whole grains for the prediction of
moisture, protein, oil, fiber and starch. Recently, the technique has been used in
attempts to distinguish RRS from conventional soybean. In this study, spectral scans
were taken from three Infratec 1220 spectrometers where whole-grain samples flow
through a fixed path length. Locally Weighed Regression using a database of ~8000
samples was 93% accurate for distinguishing RRS from unmodified soy. The
advantages of this technique are:
(1) it is fast (<1 min),
(2) sample preparation is not necessary because it uses whole kernels (~300 g),
which are dropped into measurement cells or flow through the system,
(3) it is therefore cheap.
The major disadvantage is that it does not identify compounds, thereby
necessitating a large set of samples to generate spectra. This calibration dataset is then
8
used to predict the GMO event. Thus, this method cannot be more accurate than the
reference method used to build the model. Moreover, a calibration needs to be
developed for each GMO to be predicted. Furthermore, although NIR is sensitive to
major organic compounds (e.g. vibration overtones of C---H, O---H and N---H), its
accuracy is limited. For example, with respect to GMOs, it does not detect a change in
DNA or a single protein, but much larger unknown structural changes, such as those
linked to the parietal portion of the seed (e.g. lignin or cellulose) that are introduced by
the presence of the new DNA. The procedures for detecting GMOs in foods described
here are presented in Table 6.
Table 6
3.3.2.4
Specificity of DNA-based analytical methods
PCR-based GMO tests can be categorised into four levels of specificity. The least
specific methods are commonly called "screening methods" and relate to target DNA
elements, such as promoters and terminators that are present in many different GMOs.
The second level is "gene-specific methods". These methods normally target a
part of the DNA harbouring the active gene associated with the specific genetic
modification. Examples are the Bt gene coding for a toxin acting against certain insects
or the EPSPS gene coding for tolerance against a specific herbicide. Gene-specific
methods can provide information about the traits of a present GMO, but they cannot be
used to determine whether the GMO is authorised or not, if an authorised GMO
contains the gene, because the gene can be used in several independent transformation
events. Both screening and gene-specific methods are based on detection of more or less
9
naturally occuring DNA sequences, a fact that significantly increases the risk of
obtaining false positive analytical results in tests.
The third level of specificity is "construct-specific methods", which target the
junction between two DNA elements, such as the promoter and the functional gene.
These methods target DNA sequence junctions not naturally present in nature.
However, different GMOs may share several DNA elements, for example both the same
promoter and gene, and sometimes even the same plasmid has been used to transform
plants (e.g. the two distinct maize GMOs Mon809 and Mon810).
The highest specificity is seen when the target is the unique junction found at the
integration locus between the inserted DNA and the recipient genome. These are called
"event-specific methods". Unfortunately, even the event-specific methods have their
limitations. Crossbreeding between two GMO lines may lead to so-called stacked genes.
For example, an herbicide-tolerant GMO can be combined with an insecticide-tolerant
GMO. Both sets of functional genes are present in the crossbreed but not necessarily
linked, i.e. they are likely to be situated on different chromosomes. Quantitative
methods cannot distinguish between the gene-stacked GMO and a mixture of its two
parental GMOs. This problem is only alleviated if the test is performed on material from
a single organism, such as a leaf or a single kernel of grain or seed, in which case the
presence of both target sequences is demonstrated from a single individual that
consequently must be a gene-stacked breed. In the US, this type of hybrid GMO is not
regulated if both parent GMOs are authorised. In the European Union, however, genestacked crossbreeds require separate authorisation and consequently require quantitation
as a single GMO.
3.3.3
The combined use of molecular techniques and capillary
electrophoresis in detection of transgenic foods
Genetic engineering is used in agriculture and the food industry in order to
improve:
(i) the performance of plant varieties (resistance to plagues, herbicides, and hydric
or saline stresses);
10
(ii) technological properties during storage and processing (firmness of fruits);
and/or
(iii) the sensorial and nutritional properties of food products (starch quality,
content on vitamins or essential amino acids.
Commercial use of transgenic plants and other GMOs has raised several
ideological and ethical issues in recent years. The debate is especially intense, for
several reasons, in the case of the so-called "transgenic foods" (e.g., placing on the
market food or feed consisting or containing GMOs is subject to compulsory labeling in
the European Union (EU)). Because accidental contamination of GM-material in a nonGM background is difficult to avoid, and labeling a food product as "GMO-containing"
could severely affect marketing, EU regulations have fixed a 0.9% threshold for
accidental contamination, which is not subject to any labeling requirement. This has
created a demand for analytical methods that can detect and quantify the amount of
GMO in foods.
GMOs could be detected both by PCR, for direct detection of the transgenic
DNA, or by immunological methods, for detection of the cognate proteins (limited to
tissues in which the transgene is expressed).
PCR methods are usually preferred for GMO detection because of their better
reliability and sensitivity, and because DNA is more stable than proteins.
Although RT-PCR is gaining in popularity over quantitative competitive PCR
(QC-PCR) for the quantitation of GMOs in food samples, these methods are still under
development for the simultaneous detection of several transgenes. Besides, the
interlaboratory reproducibility of RT-PCR is too low, as has been demonstrated by the
high %RSD values frequently obtained (e.g., 40% or 33.4%).
The combined use of PCR techniques and CGE seems to be a good alternative
for the detection and quantitation of transgenic organisms in foods. Thus, in the first
work published on the use of CGE to detect transgenic foods, a new CGE method was
developed to obtain reproducible separations of DNA fragments using commercially
available polymers together with bare fused silica capillaries. The method combined a
washing routine of the column with 0.1 M hydrochloride acid followed by a rinsing step
11
with solution containing 1% polyvinyl alcohol. The use of this procedure together with
a running buffer containing 2-hydroxyethyl cellulose (HEC) gave highly resolved
separations of DNA fragments in the range 80–500 bp. The separation of these DNA
fragments was achieved in ca. 20 min with efficiencies up to 1.8 × 106 plates/m and
high intra-day and inter-day reproducibility. The length up to 500 bp corresponds to the
DNA sizes more frequently amplified by PCR for detecting GMOs in foods.
In a subsequent work, some useful considerations regarding optimization of
DNA extraction from maize flour were given. Four different methods for extracting
DNA were compared and the SDS/proteinase K method was chosen as the most
convenient. DNA samples extracted from maize flour were then amplified by PCR.
To do this, a test fragment of the "foreign" cryIA(b) gene (GenBank Accession
No. I41419) was amplified using primers cryIA(b)-V3 and cryIA(b)-V4. Amplification
of a fragment of the maize starch synthase gene dull1 (a natural gene in maize), used as
a control for DNA quality and amplificability, was performed with primers MSS-S and
MSS-A. The DNA amplified by PCR was next injected into the CGE-UV equipment.
Although the sensitivity of the PCR-CGE-UV procedure was enough to detect
1% of transgenic maize in food samples, the peak obtained for the amplified DNA was
too close to the LOD, so the sensitivity of this method was maximized using CGE-LIF.
The use of LIF improves dramatically both the LOD and the linear dynamic
range obtainable in CGE compared with UV detection. Basically, there are two
procedures to supply fluorescence to DNA fragments when excited with an Ar+ laser
(usually
ex=488
nm). The first is based on covalently binding the DNA molecules with
a derivatizing agent (frequently containing fluorescein). The second uses intercalating
dyes (for double-stranded dsDNA) added to the buffer (e.g., ethidium bromide (EtBr),
thiazole orange (TO), oxazole yellow (YO), or their corresponding homodimers) that
forms stable fluorescent complexes when bound to dsDNA fragments.
Ultrasensitive detection of GM maize DNA could be achieved by CGE-LIF
using different fluorescent intercalating dyes. To do that, four different fluorescent
intercalating dyes were compared for the CGE-LIF detection of DNA from transgenic
12
maize in flours (Table 7). It was demonstrated that using YOPRO-1 as a fluorescent
intercalating dye provided optimum conditions in terms of sensitivity (i.e., LOD was
1000 zmol, calculated for a 200-bp DNA fragment), efficiency (up to 2.4 × 106
plates/m), speed of analysis, reproducibility and cost per run. Using this fluorescent
compound, the fluorescence signal was shown to vary linearly with the DNA
concentration in the range studied (i.e., 1–500 ng/ l), which was a good indication of
the quantitative possibilities of this analytical procedure. It was demonstrated that, using
this method, 0.01% of transgenic maize could be detected in flour by direct injection of
the PCR-amplified sample; Fig. 14 gives an example of this PCR-CGE-LIF analysis.
Table 7. Characteristics of four different fluorescent intercalating dyes
used to detect dsDNA by CGE-LIF using an Ar+ laser.
Figure 14. CGE-LIF electrophoregrams obtained for the PCR amplification reactions
from: (a) 0.01% transgenic maize DNA using the primer pair MSS-S/MSS-A; (b) 0.01%
transgenic maize DNA using the primer pair cryIA(b)-V5/cryIA(b)-V6; (c) conventional
13
maize DNA using the primer pair cryIA(b)-V5/c ryIA(b)-V6. Samples injected for 12 s
using N2 pressure (1 psi).
A recent work describes a new procedure useful for quantitative analysis of GMOs
in foods and applied it to analyze transgenic Bt Event-176 maize. The method
developed is based on the co-amplification of specific DNA maize sequences with
internal standards using QC-PCR. The QC-PCR products were quantitatively analyzed
using the CGE-LIF method described above. The CGE-LIF procedure allowed the use
of internal standards differing by only 10 bp from the original target fragments, which
is, to our knowledge, the smallest size difference that can be found in the literature for
QC-PCR of GMOs. It was shown that CGE-LIF provided better resolution and a
signal/noise ratio improvement of ca. 700-fold compared to SGE (Fig. 15). The good
possibilities, in terms of quantitative analysis of GMOs, provided by this new method
were confirmed by determining the Bt Event-176 maize content in different certified
reference maize powders and food samples of known composition.
Figure 15. Electrophoretic analysis of a series of QC-PCR reactions (cryIA(b) system)
with 0.21 fg of pBTIS internal standard per reaction. The analysis was performed by (a)
SGE or (b) CGE-LIF. Samples: M, DNA molecular weight marker; 1, control in the
absence of DNA template; 2, control in the absence of internal standard; 3–8,
competitive reactions with increasing amounts of transgenic Bt Event-176 maize
genomic DNA – (3) 2.5 ng, (4) 0.77 ng, (5) 0.26 ng, (6) 0.12 ng, (7) 0.04 ng, and (8) 0
ng per reaction.
14
Recently, the good possibilities of a commercial microfluidic CE system
(LabChip) have been demonstrated for the analysis of GMOs in food. It is demonstrated
that the use of microfluidic systems offer improvements in quantitative accuracy,
objectivity and ease of use compared with traditional agarose SGE.
15