Download Nuevas estrategias para la identificación de aditivos y alimentos

Nuevas estrategias para la identificación de
aditivos y alimentos
Curso de Aditivos y su Aplicación en la Industria Alimentaria
A.F.C.A.-Cámara de Comercio
Palma de Mallorca, 6-8 Marzo, 2001
Universitat de les Illes Balears and
IMEDEA (CSIC-UIB)
Area de Microbiología
Departamento de Biología y
Departamento de Recursos Naturales
Vicente J. Benedí
1
Applications of analytical molecular
biology methods. 1
• Agriculture: pathogen detection, plant breeding
programs, GMO detection, cultivar identification
• Animal husbandry: detection and treatment of infections,
genetic selection, sex identification
• Human health: genetic diseases, infectious diseases,
cancer diseases, etc.
• Environment & Ecology: Species identification, symbiotic
interactions, assessment of biodiversity
• Forensic science: individual and familial identification,
scene of crime
2
Applications of analytical molecular
biology methods. 2
• Food: pathogen detection, product/species
identification, adulteration detection, origin tracing,
GMO detection
• Law enforcement: trading standards, better
definitions, etc.
– Verify compliance with legislative requirements and
maximum permitted levels
– Confirm the authenticity of raw materials
– Improve understanding of the basic mechanisms of,
e.g., gel formation, formulations, etc.
3
DNA: the universal (biological) analyte
•
•
•
•
•
Almost universal (RNA viruses)
Extremely stable
Polymorphic and informative
Trace amounts can be amplified
Easy to work with: isolation, amplification,
detection, analysis
4
DNA and its building blocks
DNA is certainly a molecule composed only by
four blocks (or nucleotides) called A, C, G, and
T. Combinations of these four blocks give
different messages which code for all proteins
necessary for all living functions.
5
Examples of languages
(messages)
English
Musical
Score
Morse
code
Chinese
DNA
The DNA message is then a specific language.
Understanding these languages is crucial to
understanding the messages. In the DNA case,
there are some messages (sequences) that we
know they code for some proteins or functions.
6
DNA vs. amino acid information
The DNA language is certainly more
informative than other biological messages. For
example, each amino acid is coded by three
nucleotides, so we have at least three times
more information when we read the DNA
sequences than when we read the protein
sequence. Furthermore, since the DNA code is
“degenerate” (one amino acid can be coded by
more than one nucleotide triplet), there is
usually more than three times information at the
nucleotides than that at the amino acid level.
An example of the consequences of these DNA
properties can be observed in the slide.
7
DNA information is conserved
Another feature of the DNA messages that is
important for our purposes is that they read
basically the same (are conserved) among the
phylogenetic tree.
In this real example, DNA sequences
implicated in sex determination in humans and
whales are compared. Most of the sequences
from the two species are the same, as can be
expected since they code for the same
message (function). However, both species can
clearly be differentiated by specific sequence
variations.
8
DNA sequences in public databanks
(March 08, 2001)
9
The Polymerase Chain Reaction
A method based on DNA has a further
advantage over those based on other biological
molecules. By using the polymerase chain
reaction (PCR), a few DNA molecules can be
copied many times (amplified) following the
scheme shown in the slide.
For this, it is necessary and key to design two
short DNA pieces (called primers) specific for
the two extremes of the DNA sequence to be
copied.
10
Amplification step of the PCR
Once these primers have annealed to their
complementary sequences on the target DNA,
the selected sequence between the two primers
can be copied many times. Thus, from a few
molecules, one can obtain thousand of copies
of the original sequence, provided that the
primers are really specific, I.e, they anneal to
the desired two ends of the sequence to be
amplified.
11
Technical variables and detection
12
Locust bean or guar?
Molecular methods for detecting additions of
guar gum to locust bean gum
Universitat de les Illes Balears and
IMEDEA (CSIC-UIB)
Area de Microbiología
Departamento de Biología y
Departamento de Recursos Naturales
Vicente J. Benedí
This work was presented at the annual meeting
of the Institut Européen des Industries de la
Gomme de Caroube (INEC) hold in Granada,
Spain in May 200.
It describes the preexisting methods for
discriminating between the locust bean gum
(LBG)and guar gum, two gellifier agents
extracted from the carob tree (Ceratonia
siliqua) and the guar plant (Cyamopsis
tetragonolobus or C. tetragonoloba). Also, new
methods based on DNA identification are
described.
The interest in this discrimination comes from
the different prices of the two additives, the
LBG being more expensive than the guar gum.
13
• Spain is the major producer of LBG
• The Balearic Islands are the 2nd in Spain
• LBG (E 410) vs. guar (E 412) has:
• Better properties as gelling agent
• No health related problems
• Higher price
Garrofín
Guar
In this slide, we summarize the properties of
both locust bean gum (LBG) and guar gum.
Clearly, there is a major difference in price
between the two additives, thus opening the
possibilty of frauds.
14
E 410 vs. E 412: what the EC says (I. Definitions)
Off. J. of the E.C. L334, 09.12.98
E 410
Locust bean gum is the ground endosperm of the seeds of the
natural strains of carob tree, Cerationia siliqua (L.) Taub. (family
Leguminosae). Consists mainly of a high molecular weight
hydrocolloidal polysaccharide, composed of galactopyranose and
mannopyranose units combined through glycosidic linkages, which
may be described chemically as galactomannans
E 412
Guar gum is the ground endosperm of the seeds of the natural
strains of guar plant, Cyamopsis tetragonlobus (L.) Taub. (family
Leguminosae). Consists mainly of a high molecular weight
hydrocolloidal polysaccharide, composed of galactopyranose and
mannopyranose units combined through glycosidic linkages, which
may be described chemically as galactomannans
LBG and guar gum are food additives subjected
to regulations and coded as E410 and E412
respectively.
This slide shows the official definition published
in the Official Journal of the European
Communities, i.e., the official definition for most
European countries. We have copied that
definition, even with its mistake in the scientific
name of carob tree, and have highlighted in red
the differences between the two additives.
Please note that for the Official Journal, the
only differences are the plant species from
which the additives are extracted.
15
E 410 vs. E 412: what the EC says (II. Identification)
Off. J. of the E.C. L334, 09.12.98
E 410
A. Positive tests for galactose mannose
B. Microscopic examination (see next slide)
C. Solubility: soluble in hot water, insoluble in ethanol
E 412
A. Positive tests for galactose mannose
B. Solubility: soluble in cold water
Both additives are theoretically identified by the
official tests shown in this slide. It can be seen
that bot are galactomannans, i.e., polymers of
galactose and mannose, with guar gum being
more soluble in cold water than LBG.
Microscopic examination requires further
explanation, since it can be used for
differentiation. This is shown in more detail in
the next slides.
16
E 410 vs. E 412: what the EC says (II. Identification)
B. Microscopical identification
(Place some…containing 0.5% iodine and…and examine under the microscope.)
Locust bean gum contains long stretched tubiform cells,
separated or highly interspaced. Their brown contents are much
less regularly formed in guar gum. Guar gum shows close groups
of round to pear shaped cells. Their contents are yellow to
brown.
E 410
E 412
Following the official methods, LBG and guar
gum preparations can be stained and observed
under the microscope. As shown in this slide,
these two gums show clearly different
structures which can help to identify them.
17
E 410 vs. E 412: what the EC says (II. Identification)
A
Control LBG
B
Control Guar gum
Following these microscopical methods, we
studied a commercial mixture of both gums
(top panel), and, after magnification, we
identified two different structures (middle
panels). These two structures resemble those
observed for the control pure gums (bottom
panels).
However, these methods are subjective and
may require intensive observation when facing
a fraud mixture of low percentage guar in LBG.
18
Other methods from literature
• Methods based on the polysaccharide
composition
• Galactose to mannose ratios
• Methods based on the polysaccharide
composition and structure
• Lectin assays
Two other types of methods have been
described in the literature for the identification
of LBG and guar gum. Both types are base on
the polysaccharide nature of the gums, and it is
worthwhile to explain them in more detail in the
next few slides.
19
Mannose to galactose ratios (I)
• EC says nothing in E 410 and E 412
descriptions
• But when describes E 417 says that
mannose:galactose ratios are
– 3:1 for E 417
(m-m-m-m-m-m-m-m-m-m-m-m)n
I
I
I
I
g
g
g
g
– 4:1 for E 410
(m-m-m-m-m-m-m-m-m-m-m-m)n
I
I
I
g
g
g
– 2:1 for E 412
(m-m-m-m-m-m-m-m-m-m-m-m)n
I
I
I
I
I
I
g
g
g
g
g
g
The European Communities Journal does not
specify in the definitions for LBG (E410) and
guar gum (E412) the composition of these
galactomannans. However, when it defines the
tara gum (E417), a gum that was commercially
introduced later than the other two gums, the
Journal gives three different
mannose:galactose ratios for the three
polysaccharides.
20
Mannose to galactose ratios (II)
• 4:1 for E 410; 2:1 for E 412
• But, different ratios have been described:
– 3.0:1 and 1.5:1 (Preuss and Their, Z Lebensm Unters Forsch, 175:93, 1982)
– 2.7:1 and 1.4:1 (Angelini et al, Riv Soc Ital Alim, 13:479, 1984)
– 3.7:1 and 2.3:1 (Cheetham et al, Carbohyd Pol 6:257, 1986)
– 3.7 to 7.7:1 depending on the solubilization
temperature of E 410 (Lopes da Silva and Gonzales, Foof Hydrocol. 4:277, 1990)
However, these differences cannot be exploited
for the differentiation of LBG and guar gum,
since the literature describes variations in the
galactose to mannose ratios. It would still be
possible, for isolated gums, to ensure if the
analyzed sample is LBG or guar because for
LBG these ratios are always higher than for
guar gum. However, in a fraud situation, when
a theoretically pure LBG sample may contain
5% guar, these variations in the galactose to
mannose ratio make impossible to ensure the
presence of guar.
21
Mannose to galactose ratios (III)
• Variations difficult demonstration of E 412
presence in E 410/E 412 mixtures
• Furthermore, man:gal ratios will be affected if
other food additives (e.g., man from E 415)
• Also, man and gal can be present in processed
foods, thus affecting the man:gal ratios
Techniques are cumbersome and require complex extractions
Methods based on the galactose to mannose
ratio are further limited by other considerations.
First, the food industry often use combinations
of additives. So for example, the presence of
additive E415 will affect the ratio due to the
mannose of this additive. Also, mannose and
galactose are present in many processed
foods, thus affecting the galactose to mannose
ratio of the food additives. Finally, the methods
required to study galactose to mannose ratios
are cumbersome and require complex
extractions and derivatizations.
22
Lectin assays
• Lectins are plant proteins which bind polysaccharides
• Different lectins bind different polysaccharides
• Patel et al. (Leatherhead Food RA)
LBG
lectin
enzyme
guar
GUAR
support
immobilized
lectin
LBG
lectin binds guar
but not LBG
bound guar is
detected with the
same lectin
labeled with an
enzyme
color
readings
A second type of method described by Patel
and colleagues use lectins. This plant-derived
proteins bind some polysaccharides through
specific interactions between a given lectin and
a polysaccharide sequence. It has been
described that one of such lectins can be used
in an ELISA-type method to detect guar gums,
since it will bind guar and not LBG.
The principles of the method is shown on this
slide.
23
Enzyme-Linked Lectin Assay (ELLA)
1.2
Optical density
1.0
0.8
0.6
0.4
0.2
0
LBG
Guar
Tara
E 410
commercial samples
buffer
However, when we used the quantitative lectin-based
methods (called ELLA by the original authors) we
found the results shown in this slide. Certainly, the
ELLA method, when applied to pure control
preparations of LBG (yellow bars) and guar gum (red
bars) is able to differentiate the by their different
reactivity with the enzyme-labeled lectin, with LBG
being more reactive. The tara gum (blue bars) gave
values between those of the two other gums.
However, when this method was applied to
commercial preparations labeled as “LBG”, we
obtained a whole range of reactivities, and for many
of these samples it was no possible to rule out the
possible presence of guar gum.
24
Development of new (DNA-based) methods
E 410
Locust bean gum is the ground endosperm of the seeds of the
natural strains of carob tree, Cerationia siliqua (L.) Taub. (family
Leguminosae). Consists mainly of a high molecular weight
hydrocolloidal polysaccharide, composed of galactopyranose and
mannopyranose units combined through glycosidic linkages, which
may be described chemically as galactomannans
E 412
Guar gum is the ground endosperm of the seeds of the natural
strains of guar plant, Cyamopsis tetragonlobus (L.) Taub. (family
Leguminosae). Consists mainly of a high molecular weight
hydrocolloidal polysaccharide, composed of galactopyranose and
mannopyranose units combined through glycosidic linkages, which
may be described chemically as galactomannans
We then decided to develop a new method for
the reliable detection and differentiation of guar
gum and LBG. This method will be based on:
• The fact that the gums are extracted
from two plants of different genuses and
species.
• DNA sequences which could be specific
from these two plants
25
Seeds as sources of DNA
• Germination of seeds (carob and guar)
• Extraction of DNA from fresh tissues
• PCR amplification of extracted DNA
• Electrophoretic analysis of amplification products
G
U
A
R
L
B
G
Marker
1
G
U
A
R
L
B
G
Marker
2
On germinated seeds of the guar plant and
carob tree, we have found two DNA regions
that we called “markers” because they exist in
both plants but (as seen in the next slides) with
specific differences in their sequences
depending on the species.
26
Unveiling the Markers sequences
• Isolation of markers from gel
• Sequencing
L
B
G
G
U
A
R
Marker
1
L
B
G
G
U
A
R
Marker
2
GUAR sequence (Marker 1)
ACCTTCCTCTTCAGCATTGTTCCAAAGGCATCCACTTGGACGCCTTCCTAGTAACAG
CTACGGAGTGTTCGTCAGGCTGGGCACTTGAACAAAACGAATAAATCCCAACCAAAC
CCCGCACAGTTTTGTGCGGCTGGAAGGAAACCAACCCTCAACAGACGGAACGCACCG
AAAGAGAATCGGAAATTGTTTGGGTGGCCGCGATGTGCGCGGTTCCTTTGAATTGAN
AAGACACGCGGGAACGGTCGGGCCATTGCCACGACACATCCAACNCAAATCTATGTA
CTTAGTTTTACTGAGAGCCGTTGCCTATAGAGCCGAGAGCGTAGCTACTTCTTGCGT
CGT
CAROB TREE sequence (Marker 1)
ACCTTCCTCTTCAGCATTGTTCCAAAAGCATCCACTTGGACACCTTCCTAGTAACAG
CTACGGAGTGTTTTGCTTGCTGGACGCTTAACCAATTTGATAGCCCCCGCCCCCCGC
ACGCAGGAGGGTTCGGAGGTACAGCCCTCCGCGGACACCGGGGGGCGGTGAGCACGA
TGGAGCTGGTTTTTTGATTGGGACCGCAAATTGCGCGGTTCCTTGATGTTGGTCACT
CGCACGAGGGCTACTGGACCATTGCCGCTAGCTAGCTACTCGCAGCACTGTAAGAAT
AGGTTTTACTGAGAGCCATTGCCTATAGAGCCGAGAGCGTAGCTACTTCTTGCGTCG
T
These differences could be demonstrated by
isolating the markers and sequencing them.
The slide shows underlined one of these
differences in sequence found in the markers of
the two plants.
27
Marker 1 restriction analysis
G
L UL
B A B
G RG
G
U
A
R
G
L U
B A
G R
ClaI
HaeIII
PCR amplification
and digestion
BcnI
Additionally, the differences in sequence
between the markers of the two plants can be
easily characterized by their differential
susceptibility to restriction endonuclesases
(enzymes which cleave specific DNA
sequences).
As shown in the slides, the two plants markers
were clearly different by this type of analysis.
28
Marker 2 restriction analysis
G
G
L UL U
B A B A
G RG R
G
L U
B A
G R
SmaI XhoI HaeIII
This as another example of the same type of
analysis but applied to the second marker.
29
Markers sequences
• Isolation of markers from gel
• Sequencing
L
B
G
G
U
A
R
Marker
1
L
B
G
G
U
A
R
Marker
2
GUAR sequence (Marker 1)
ACCTTCCTCTTCAGCATTGTTCCAAAGGCATCCACTTGGACGCCTTCCTAGTAACAG
CTACGGAGTGTTCGTCAGGCTGGGCACTTGAACAAAACGAATAAATCCCAACCAAAC
CCCGCACAGTTTTGTGCGGCTGGAAGGAAACCAACCCTCAACAGACGGAACGCACCG
AAAGAGAATCGGAAATTGTTTGGGTGGCCGCGATGTGCGCGGTTCCTTTGAATTGAN
AAGACACGCGGGAACGGTCGGGCCATTGCCACGACACATCCAACNCAAATCTATGTA
CTTAGTTTTACTGAGAGCCGTTGCCTATAGAGCCGAGAGCGTAGCTACTTCTTGCGT
CGT
CAROB TREE sequence (Marker 1)
ACCTTCCTCTTCAGCATTGTTCCAAAAGCATCCACTTGGACACCTTCCTAGTAACAG
CTACGGAGTGTTTTGCTTGCTGGACGCTTAACCAATTTGATAGCCCCCGCCCCCCGC
ACGCAGGAGGGTTCGGAGGTACAGCCCTCCGCGGACACCGGGGGGCGGTGAGCACGA
TGGAGCTGGTTTTTTGATTGGGACCGCAAATTGCGCGGTTCCTTGATGTTGGTCACT
CGCACGAGGGCTACTGGACCATTGCCGCTAGCTAGCTACTCGCAGCACTGTAAGAAT
AGGTTTTACTGAGAGCCATTGCCTATAGAGCCGAGAGCGTAGCTACTTCTTGCGTCG
T
Furthermore, as we mentioned a few slides
before, the markers from the two plants differ in
their sequences, and one can exploit these
differences to develop highly specific methods
for the detection and differentiation of the two
species.
30
Carob vs. guar sequences (Marker 2)
GUAR
CAROB
different
P3
identical
1 123413241244244323423 2444123 2414311221212441123424
1 1234132412442443234232444123 4414311221212441123424
50
50
GUAR
CAROB
51 413 332324433 113242131112443 23442112 3233324423341 14 100
51 413 112124433 413442131112443 43442112 1233324423341 34 100
GUAR
CAROB
PG22
101 22332422234323312331222 3213 2323213 2112333 14431322- 149
101 42332442234321312331222 1213 4343431 2132333 334333321 150
GUAR
CAROB
150 -4321313 411 122 12132 11242122 -32441 211223 11333432423 197
151 22331313 232 122 31132 42244122 234241 141223 31133212423 200
GUAR
CAROB
198 2112331 34122 1122 112 444 113 2421 332122122 4224 12334324 247
201 1132331 14122 4122 333 444 424 2421 132322122 3243 12334324 250
GUAR
CAROB
PG21
248 11241 4122122 11242 144 1142313 242433 3413212 423 24313-3 296
251 3--23 4322122 41242 344 424-313 442433 4213212 344 2121341 297
GUAR
CAROB
PG21
297 41311211112 24313 111 24333 nnnn4 341 2423 413133 2-4123 13 341
298 43332224112 32313 224 24333 11342 341 3232 422123 414123 31 347
GUAR
CAROB
P4
342 211 432 42433 134221342 432222 1143 33231242111442341413
348 324 432 22433 314421342 232222 3143 13231242111442341413
GUAR
CAROB
P4
392 4414423224224
398 4414423224224
391
397
405
410
In this slide we show a schematic comparison
of the sequences of the marker 2 sequences
from the carob tree and guar plant. Most of
these sequences coincide (yellow regions) as
could be expected from the fact that they code
for the same function. Other zones (in red) are
clearly different between the two species, just
as it happened in the whale vs. human example
shown some slides before.
These sequence differences were used to
design primers P3 and P4 which amplify both
species, as well as primers PG21 and PG22
that are specific of the guar DNA.
31
Specific amplification of guar DNA from seeds
400
300
200
100
Guar seeds
Carob tree seeds
By using the specific guar primers, we
demonstrated that DNA extracted from guar
seeds was detected after amplification,
whereas no amplification was detected with the
same primers and the DNA extracted from
carob tree seeds.
32
Investigation of DNA extraction from locust bean and
guar gums (E 410 and E 412)
Gum
Guar
30% Guar
10% Guar
Locust bean
Guar
30% Guar
10% Guar
Locust bean
Guar
30% Guar
10% Guar
Locust bean
Guar
30% Guar
10% Guar
Locust bean
Extraction
method
1
1
1
1
2
2
2
2
3
3
3
3
4
4
4
4
DNA
Sugars
DNA/sugars
PCR
46.87
8.6
10.8
14.22
77.3
156.1
232.5
509.7
4.84
1.72
1.94
1.72
3.21
0.3
0.64
0.86
0.639
0.523
0.541
0.572
1.056
0.361
0.494
0.721
0.013
0.019
0.030
0.029
ND
ND
ND
ND
1 / 14,000
1 / 60,000
1 / 50,000
1 / 40,000
1 / 14,000
1 / 2,500
1 / 2,000
1 / 1,500
1 / 3,000
1 / 11,000
1 / 16,000
1 / 17,000
NA
NA
NA
NA
+
+
+
—
+
—
—
—
+
—
—
—
—
—
—
—
The purpose of the method was to differentiate
the two gums, not just the two seeds.
Commercial gums extracted from the seeds of
guar plan t and carob tree contain DNA that can
be amplified by PCR and the guar-specific
primers. However, a critical step in the method
was to find out an extraction procedure of the
DNA from the galactomannan matrix. This slide
shows some of the assayed extraction
methods. It can clearly be seen that only
extraction method 1 was suitable for further
PCR amplification, although this extraction
procedure was not the most effective, in
quantitative terms, for DNA extraction.
33
Specific amplification of guar DNA from mixtures of LBG and
known additions of guar gum
300
200
100
L
B
G
G
U
A
R
30% 20% 10% 12% 6%
mixture 1
2% guar+LBG
mixture 2
Using the right extraction procedure and guarspecific primers, we amplified and detected the
presence of guar gum in laboratory control
mixtures of LBG and guar gum.
34
Specific detection of guar DNA in
commercial samples labeled as E 410
LBG
300
Guar
200
100
Commercial samples
labeled as E 410
When these methods were applied to the
analysis of commercial samples of locust bean
gum, we in fact detected the presence of guar
in some of these theoretically pure “LBG”.
However, it should be emphasized that the
number of samples positive for guar does not
represent the real proportion of positives we
have found in our survey of market samples.
35
Specificity of the amplified products
Amplification products were
• cutted with a restrictase specific of the guar sequence
• analyzed by gel electrophoresis
uncut
Taq I
XhoI
restrictions
Further proof of the specificity of the detected
guar was obtained by restriction analysis of the
amplicon. This slides shows that the amplified
product gave the restriction fragments expected
for guar DNA.
36
100% Guar
100% LBG
90% LBG-10%Guar
Fluorescence detection of guar amplicons in guar guma and
LBG-guar gum mixtures. Both the molecular size and the
abundance of guar amplicons can be detected in a few
minutes
37
Molecular methods for detecting additions of
guar gum to locust bean gum
Work developed by:
• S. Albertí, A. Doménech-Sánchez, V.J. Benedí
• M.L. Hernández, J.A. Rosselló
With the collaboration of:
• A. Juan, J. Sansegundo (Carob S.A., lab)
• D. Álvarez, M. Urdiaín (IMEDEA, CSIC-UIB)
Funded by:
In summary, using DNA sequences specific
from carob tree and guar plant, we have
developed a method for the PCR amplification
of specific guar sequences. The presence of
these sequences in LBG-guar gum mixtures
can be detected by PC R using specific guar
primers.
The method is patent pending, and the people
and Institutions cited in the slide have
contributed to its development.
38
Las nuevas nuevas tecnologías
Qué es lo que está llegando?
39
Real-time PCR (quantitative)
In a conventional PCR, the number of amplicons increases as the
number of cycles increases, but it is not until that a sufficient number of
cycles have ben run that the amplicons can be detected (see top of the
figure).
A conventional PCR is usually performed for 35-45 cycles and is
basically an “end-point” analysis: two samples with two different
amounts of target DNA to be amplified are PCR amplified for the same
number of cycles and the compared by agarose gel electrophoresis. It
could be possible to estimate the amount of amplicons in two samples
by removing samples at different cycles, running them in an agarose
gel, and then comparing the intensity of the bands from the gel. This is
not done because it is cumbersome and increases the chances of
contamination of the PCR mixtures.
The real-time PCR machine provides real-time monitoring of the
amount of new amplicons formed troughout every cycle. The reasons
are: (1) a fluorecent dye is incorporated in the PCR mixture, and (2) the
fluoresce in the mixture is read by buil-in fluorescence detectors.
40
The real-time PCR
machine
The real-time PCR machine, like the LightCycler (Roche)
shown in the slide is a PCR machine which works with
capilars instead that with tubes. This allows faster
temperature exchange between the PCR mixture and the
cooling device, thus making the PCR cycles to become faster
(shorter). An important feature of these real-time PCR
machines is that fluorescence in the capilars is constatntly
monitored, thus informing of the amount of amplicons being
formed in every cycle of the PCR.
41
SYBR Green fluorescent dye
does not fluoresce until binding
to double strand DNA
At the beginning of amplification, the reaction
mixture contains the denatured DNA, the primers,
and the dye. The unbound dye molecules (SYBR
Green, Molecular Probes) weakly fluoresce,
producing a minimal background fluorescence
signal which is subtracted during computer
analysis.
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Upon binding primers, the dye
starts to fluoresce
After annealing of the primers, a few dye molecules can
bind to the double strand. DNA binding results in a
dramatic increase of the SYBR Green I molecules to emit
light upon excitation.
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As more and more double strand DNA
molecules (amplicons) are formed, more
SYBR green molecules bind and
fluorescence increases
During elongation, more and more dye molecules bind to
the newly synthesized DNA. If the reaction is monitored
continuously, an increase in fluorescence is viewed in
real-time. Upon denaturation of the DNA for the next
heating cycle, the dye molecules are released and the
fluorescence signal falls.
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Different PCR curves are obtained for different amounts of initial
DNA. End-points (plateaus) are obtained at different cycles: those
samples with higheramounts of initial target DNA reach the plateau
more rapidly
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102 103 104 105
Real-time quantitative PCR analysis
a) Analysis of the amplification plots for the standard
concentrations (in duplicate) of the target DNA
b) Standard curve; plot of the crossing point (cycle number)
against the input target quantity
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