 Dyes & Fluorescence detection chemistry in qPCR
by Ambion TechNotes 8(1) - February 2001 http://www.ambion.com/techlib/tn/81/813.html All real-time PCR systems rely upon the detection and quantitation of a fluorescent reporter, the signal of which increases in direct proportion to the amount of PCR product in a reaction. In the simplest and most economical format, that reporter is the double-strand DNA-specific dye SYBR® Green (Molecular Probes). SYBR Green binds double-stranded DNA, and upon excitation emits light. Thus, as a PCR product accumulates, fluorescence increases. The advantages of SYBR Green are that it's inexpensive, easy to use, and sensitive. The disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which results in an overestimation of the target concentration. For single PCR product reactions with well designed primers, SYBR Green can work extremely well, with spurious non-specific background only showing up in very late cycles. The two most popular alternatives to SYBR Green are TaqMan® and molecular beacons, both of which are hybridization probes relying on fluorescence resonance energy transfer (FRET) for quantitation. TaqMan Probes are oligonucleotides that contain a fluorescent dye, typically on the 5' base, and a quenching dye, typically located on the 3' base. When irradiated, the excited fluorescent dye transfers energy to the nearby quenching dye molecule rather than fluorescing, resulting in a nonfluorescent substrate. TaqMan probes are designed to hybridize to an internal region of a PCR product. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5' exonuclease activity of the polymerase cleaves the probe. This separates the fluorescent and quenching dyes and FRET no longer occurs. Fluorescence increases in each cycle, proportional to the rate of probe cleavage. Molecular beacons also contain fluorescent and quenching dyes, but FRET only occurs when the quenching dye is directly adjacent to the fluorescent dye. Molecular beacons are designed to adopt a hairpin structure while free in solution, bringing the fluorescent dye and quencher in close proximity. When a molecular beacon hybridizes to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. Unlike TaqMan probes, molecular beacons are designed to remain intact during the amplification reaction, and must rebind to target in every cycle for signal measurement.
Randy Rasmussen
Ph.D. (Idaho Technology, Chief Operating Officer) SYBR Green I is
dsDNA-binding dye. It is thought to bind in the minor groove of
dsDNA and
upon binding increases in fluorescence over a hundred fold (Figure
8a). It is compatible with PCR up to a point, at very high
concentrations
it starts to inhibit the PCR reaction. In the LightCycler
Instrument, SYBR is monitored in channel F1. The biggest advantage of
SYBR is that
it binds to any dsDNA; there is no designing and optimizing of probes
required. If you have a PCR that works, you can have a real-time
quantitative assay working in about a day. Hybridization Probes If sequence
specific recognition is required, the HybProbe system allows detection
of only the specific product. Two probes are designed that
hybridize side by side on the PCR product (Figure 8c). The 3’ end
of the upstream probe is labeled with fluorescein, which acts as a
fluorescence resonance energy transfer (FRET) donor. The 5’ end
of the downstream probe is labeled with an acceptor dye, either LC Red
640, or LC Red 705. The FRET signal is seen only when two
specific hybridization events occur. In the LightCycler
Instrument, LC Red 640 is monitored in channel F2, LC Red 705 in
channel F3. There may sometimes be an advantage to monitoring the
ration of the acceptor channel (where the signal goes up with
increasing PCR product) and the signal from fluorescein in F1 (which
goes down with increasing PCR product.
<> TaqMan® Probes TaqMan probes
derive their fluorescence signal from the hydrolysis of the probe by
Taq’s 5’ to 3’ exonuclease activity (Figure 8c). The hydrolysis
separates fluorescein from a quenching dye and results in an increased
fluorescein signal. These probes can be used in the LightCycler
Instrument and are monitored in F1 or F1/F2.
The fluorescent
dye SYBR Green I binds to the minor groove of the DNA double helix. In
solution, the unbound dye exhibits very little fluorescence, however,
fluorescence is greatly enhanced upon DNA-binding. Since SYBR Green I
dye is very
stable (only 6% of the activity is lost during 30 amplification cycles)
and the LightCycler instrument's optical filter set matches the
wavelengths of excitation and emission, it is the reagent of choice
when measuring
total DNA. The principle is outlined in the following figures.
At the beginning
of amplification, the reaction mixture contains the denatured DNA, the
primers, and the dye. The unbound dye molecules weakly fluoresce,
producing a minimal background fluorescence signal which is subtracted
during computer analysis.
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.
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. Fluorescence
measurement at the end of the elongation step of every PCR cycle is
performed
to monitor the increasing amount of amplified DNA. Together with a
melting curve analysis performed subsequently to the PCR, the SYBR
Green
I format provides an excellent tool for specific product identification
and quantification. Demonstration of preferential binding of SYBR
Green I to specific DNA fragments Steven
Giglio*, Paul T. Monis and Christopher P. Saint Nucleic Acids Research, 2003, Vol. 31, No. 22 e136  SYBR Green I (SG) is widely used
in real-time PCR applications as an intercalating dye and is included in many commercially available
kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-speciÆc and
additional testing, such as DNA melting curve analysis, is required to
conÆrm the generation of a speciÆc
amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis
because the melting temperature (Tm) of the speciÆc amplicon is analogous to the detection of an
electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of
amplicons should be possible, provided the Tm values are suffiently different. Real-time multiplex
assays for Vibrio cholerae and
Legionella pneumophila using commercially available kits and in-house SG
mastermixes have highlighted variability in performance characteristics, in particular the
detection of only a single
product as assessed by Tm analysis but multiple products as assessed by
agarose gel electrophoresis.
The detected Tm corresponds to the amplicon with the higher G+C%
and larger size, suggesting
preferential binding of SG during PCR and resulting in the failure to
detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This
has implications for the design and routine application of diagnostic real-time PCR assays employing SG.
A
new minor groove binding asymmetric cyanine reporter dye for real-time
PCR
Martin Bengtsson, H. Jonas Karlsson, Gunnar Westman and Mikael Kubista* Department of Chemistry and Bioscience, Chalmers University of Technology 41296 Go»teborg and TATAA Biocenter, Medicinaregatan 9E, 413 90 Goteborg, Sweden Nucleic Acids Research, 2003, Vol. 31, No. 8 e45 The minor groove binding
asymmetric cyanine dye 4-[(3-methyl-6- (benzothiazol-2-yl)-
2,3-dihydro- (benzo-1,3-thiazole)
-2-methylidene)]- 1-methyl-pyridinium iodide (BEBO) is tested as
sequence nonspeciÆc label in real-time PCR. The
Fluorescence intensity of BEBO increases upon binding to double-stranded DNA allowing
emission to be measured at the end of the elongation phase in the PCR cycle. BEBO concentrations
between 0.1 and 0.4 mM generated sufÆcient Øuorescence
signal
without inhibiting the PCR. A comparison with the commonly used reporter dye SYBR Green I shows that the two dyes behave similarly in all important aspects. BEBO
for qPCR and HRM
TATAA Biocenter
AB, Medicinaregatan 8A, 413 46 Göteborg, Sweden
BEBO is an unsymmetric cyanine dye developed by TATAA Biocenter for use in qPCR applications. The dye has absorbance and emission wavelengths that can be detected on the FAM channel on most common real-time PCR platforms, and shows a strong fluorescence increase when bound to dsDNA. BEBO can be used as an unspecific dye for real-time PCR applications or other applications where staining of dsDNA is wanted. Combining sequence-specific probes and DNA binding dyes in real-time PCR for Currently,
in real-time PCR, one often has to choose between using a sequence-specific
probe and a nonspecific double-stranded DNA (dsDNA) binding dye
for the detection of amplified DNA products. The sequence-specific
probe has the advantage that it
only detects the
targeted product, while the nonspecific dye has the
advantage that melting curve analysis can be performed after completed
amplification, which reveals what kind of
products have been formed. Here we present a new
strategy based on combining a sequence-specific probe and a
nonspecific dye, BOXTO, in the same reaction, to take the advantage of
both chemistries.
We show that BOXTO can be used
together with both TaqMan probes and locked nucleic
acid (LNA) probes without interfering with the PCR. The probe signal
reflect formation of target product, while melting curve analysis of
the BOXTO
signal reveals primer-dimer formation and the
presence of any other anomalous products.
BioTechniques - BioSpotlight: Think outside the BOXTO Info about BOXTO on the TATAA Biocenter web page BOXTO as a real-time thermal cycling reporter dye ASHRAF I AHMAD Journal of Biosciences, Volume 32, Number 2 / March, 2007 The unsymmetrical cyanine dyes BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro- (benzo-1,3-thiazole)-2-methylidene)]- 1-methyl-quinolinium chloride) and its positive divalent derivative BOXTO-PRO (4-[3-methyl-6-(benzoxazole-2-yl)- 2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]- 1-(3-trimethylammonium-propyl)- quinolinium dibromide) were studied as real-time PCR reporting fluorescent dyes and compared to SYBR GREEN I (SG) (2-[N-(3-dimethylaminopropyl)-N-propylamino]- 4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]- 1-phenylquinolinium). Unmodified BOXTO showed no inhibitory effects on real-time PCR, while BOXTO-PRO showed complete inhibition, Sufficient fluorescent signal was acquired when 0.5–1.0 µM BOXTO was used with RotorGene and iCycler platforms. Statistical analysis showed that there is no significant difference between the efficiency and dynamic range of BOXTO and SG. BOXTO stock solution (1.5 mM) was stable at −20°C for more than one year and 40 µM BOXTO solution was more stable than 5x SG when both were stored at 4°C for 45 days. (the ABI TaqMan Probes) Real-time systems for PCR were improved by probe-based, rather than intercalator-based, PCR product detection. The principal drawback to intercalator-based detection of PCR product accumulation is that both specific and nonspecific products generate signal. An alternative method, the 5' nuclease assay, provides a real-time method for detecting only specific amplification products. During amplification, annealing of the probe to its target sequence generates a substrate that is cleaved by the 5' nuclease activity of Taq DNA polymerase when the enzyme extends from an upstream primer into the region of the probe. This dependence on polymerization ensures that cleavage of the probe occurs only if the target sequence is being amplified. The development of fluorogenic probes made it possible to eliminate post-PCR processing for the analysis of probe degradation. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by Förster resonance energy transfer (FRET) through space. Probe design and synthesis has been simplified by the finding that adequate quenching is observed for probes with the reporter at the 5' end and the quencher at the 3' end. Figure 1 diagrams what happens to a fluorogenic probe during the extension phase of PCR. If the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5' nuclease activity of Taq DNA polymerase as this primer is extended. This cleavage of the probe separates the reporter dye from quencher dye, increasing the reporter dye signal. Cleavage removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process. Additional reporter dye molecules are cleaved from their respective probes with each cycle, effecting an increase in fluorescence intensity proportional to the amount of amplicon produced. The
advantage of fluorogenic probes over DNA binding dyes is that specific
hybridization between probe and target is required to generate
fluorescent signal. Thus, with fluorogenic probes, non-specific
amplification due to mis-priming or primer-dimer artifact does not
generate signal. Another advantage of fluorogenic probes is that they
can be labeled with different, distinguishable reporter dyes. By using
probes labeled with different reporters, amplification of two distinct
sequences can be detected in a single PCR reaction. The disadvantage of
fluorogenic probes is that different probes must be synthesized to
detect different sequences.
The detection principle of LC™ Hybridization Probes (HybProbes) is Fluorescence Resonance Energy Transfer (FRET), the phenomenon of energy transfer from a donor to an acceptor fluorophor. If the donor and the acceptor fluorophor are in close proximity to each other, excitation of the donor by blue light results in energy transfer to the acceptor, which can then emit light of longer wavelength. This fact forms the basis for Roche’s real-time online LightCycler™ PCR System. It allows formation of PCR products to be monitored by using two sequence specific, fluorescent labeled oligonucleotide probes, called Hybridization Probes, in addition to the PCR primers. For this LC™
real-time PCR detection format the following are the major steps:
HybProbes are designed as a pair of which one probe is labeled with the donor (3´Fluo) and one with the acceptor (5´ LCRed 640 or LCRed 705) dye. As FRET decreases with the sixth power of distance, HybProbes have to be designed to hybridise to adjacent regions of the template DNA (separated by 1-5 nucleotides). If both probes hybridise, the two dyes are brought close together and FRET to the acceptor dye results in a signal measurable by the built-in fluorimeter of the LightCycler™. The fluorescence signal disappears by increasing temperature above the melting temperature of the oligos because the probes melt away from the template strand which significantly increases the distance between the dyes. Mismatches
between the
probes and the target decrease the melting temperature of the
respective probe
compared to a perfectly matched probe. This effect can also be used to
detect
SNPs by melting curve analysis.
PCR Monitoring
with Hybridization Probes The Hybridization
Probe format is used for DNA detection and quantification and provides
a maximal specificity for product identification. In addition to the
reaction components used for conventional PCR, two specially designed,
sequence specific oligonucleotides labeled with fluorescent dyes are
applied
for this detection method. This allows highly specific detection of the
amplification product as described below.
The first dye (fluorescein) is excited by the LightCycler's LED (Light Emitting Diode) filtered light source, and emits green fluorescent light at a slightly longer wavelength (middle figure). When the two dyes are in close proximity (as shown in the lower figure), the emitted energy excites the LC Red 640 attached to the second hybridization probe that subsequently emits red fluorescent light at an even longer wavelength. This energy transfer, referred to as FRET (Fluorescence Resonance Energy Transfer) is highly dependent on the spacing between the two dye molecules. Only if the molecules are in close proximity (a distance between 1–5 nucleotides) is the energy transferred at high efficiency. Choosing the appropriate detection channel, the intensity of the light emitted by the LightCycler – Red 640 is filtered and measured by the LightCycler instrument's optics. The increasing
amount of measured fluorescence is proportional to the increasing
amount of DNA generated during the ongoing PCR process. Since LC Red
640 only emits a signal when both oligonucleotides are hybridized, the
fluorescence measurement is performed after the annealing step.
Hybridization probes can be labeled with LightCycler – Red 640 and with
LightCycler – Red 705.
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Hybridization Probes for the Detection of Nucleic Acids in Homogeneous Solutions Department of
Molecular Genetics, Public Health Research Institute
Table of contents: When You Wish Upon A Star: Molecular Beacons: Real Time in a Twinkle by Deborah Wilkinson http://www.the-scientist.com/yr1999/mar/profile3_990301.html
Prime and Shine While Saving Time: Intergen's Amplifluor allows Direct Detection of PCR Products by Deborah Wilkinson http://www.the-scientist.com/yr1999/august/tools2_990830.html
Table of Licensed Providers of
Molecular
Beacons and Kits 
http://www.synthegen.com/ Specializing in Modified Oligonucleotides
SYNTHEGEN specializes exclusively in modified oligonucleotides.
Differently-colored
molecular probes specific for the wild-type and mutant alleles are
designed. DNA amplified from homozygous wild-type individuals binds
only to the fluorescein-labeled molecular beacons (left). DNA from
homozygous mutants binds only the tetramethylrhodamine-labeled
molecular beacons (right). Both types of molecular probes will bind to
amplicons generated from the DNA of heterozygous individuals
(center).
by L.G.
Kostrikis et al. (1998)
Our genotyping
process is based on Scorpions Technology - a homogeneous or closed tube
method with a simple mix and glow operation. A DNA sample is added to a
Scorpions test and an increase in fluorescence indicates the genotype.
There is no post-PCR manipulation and the use of two fluorescent dyes
gives single tube SNP analysis
Scorpions is a
class leading PCR detection technology with significant benefits
Scorpions
are bi-functional molecules containing a PCR
primer element covalently linked to a probe
element. The molecules also contain a fluorophore that
can interact with a quencher to reduce fluorescence. When the molecules
are
used in a PCR reaction the fluorophore and the quencher are separated
which
leads to an increase in light output from the reaction tube.
The benefits of Scorpions derive from the fact that the probe element is physically coupled to the primer element - this means that the reaction leading to signal generation is a uni-molecular rearrangement. This contrasts to the bi-molecular collisions required by other technologies such as Taqman or Molecular Beacons. The benefits of a
uni-molecular rearrangement are significant - as the reaction is
effectively instantaneous it occurs prior to any competing or side
reactions such as target
amplicon re-annealing or inappropriate target folding. This leads
to stronger signals, more reliable probe design, shorter reaction times
and better discrimination.
The presence of the blocker group is an essential element of the Scorpions invention. Without such a blocker the Taq DNA polymerase would be able to read through the Scorpions primer and copy the probe region. This would generate signal but not in a target specific fashion. Copying the tail in this way would completely negate the benefits of the Scorpions reaction as any inappropriate side-reactions, including the formation of primer dimers, would also generate a signal. Scorpions are PCR
primers with a " Stem-Loop " tail containing a fluorophore and a
quencher
(figue1).
Whitcombe, D., Theaker J., Guy, S.P., Brown, T., Little, S. (1999) Nature 17, 804-807
Molecular
diagnostics is progressing from low-throughput, heterogeneous, mostly
manual technologies to higher throughput, closed-tube, and automated methods.
Fluorescence is the favored signaling technology for such assays,
and a
number of techniques rely on energy transfer between a fluorophore
and a proximal
quencher molecule. In these methods, dual-labeled probes hybridize
to an amplicon and changes in the quenching of
the fluorophore are detected. We describe a new technology that is
simple to use,
gives highly specific information, and avoids the major difficulties of
the alternative methods. It uses a primer with an integral tail
that is used to probe an extension product of the primer. The probing
of a target sequence
is thereby converted into a unimolecular event, which has substantial
benefits in terms of kinetics, thermodynamics, assay design, and probe
reliability.
Design considerations and effects of LNA
in PCR primers
David Latorra1, Khalil Arar*, J. Michael Hurley2 Proligo LLC, 6200 Lookout Road, Boulder, CO 80301, USA Molecular and Cellular Probes 17 (2003) 253–259
The
effects of comprehensive LNA substitution in PCR primers for
amplification of human genomic DNA targets are presented in this
report. Previous research with LNA in other applications has shown
interesting properties for molecular hybridization including enhanced
specificity in allele-specific PCR.
Here we systematically modified PCR primers and conditions for the
human
genomic DNA targets APOB and PAH, along with a b-globin amplification
control, to study whether the number and position of LNA residues
improves
or diminishes amplification sensitivity and specificity. It was
observed
that the design rules for LNA substitution in PCR primers are complex
and depend upon number, position and sequence context. Technical
advantages
were seen when compared to DNA controls for the best LNA primer
designs,
which were typically one to a few centrally located LNA residues. LNA
advantages include increased maximum annealing temperature
ðTmaxÞ
and increased signal with limiting primer or Taq DNA polymerase.
Several
well-characterized designs exhibited different efficiencies with
different
brands of hot-start enzymes. Many shorter LNA primers were found to be
functional compared to same-length non-functional native DNA controls.
These results show that LNA-substituted PCR primers have potential for
use in difficult PCR techniques, such as multiplex amplification at
higher
Tmax; once firm LNA primer design rules are established.
A new class of homogeneous nucleic acid probes based on specific displacement
The Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of We have developed a new class of
probes for homogeneous nucleic acid detection based on the proposed displacement
hybridization. Our probes consist of two complementary
oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in
close proximity in the duplex. The probes on their own are quenched, but they become
fluorescent upon displacement hybridization with the target. These probes
display complete discrimination between a perfectly matched target and
single nucleotide mismatch targets. A comparison of double-stranded probes with
corresponding linear probes confirms that the
presence of the complementary strand significantly enhances their specificity. Using four such probes labeled with different color fluorophores, each designed to recognize a different target, we have demonstrated that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. Double-stranded probes were used in real-time nucleic acid amplifications as either probes or as primers. In addition to its extreme specificity and flexibility, the new class of probes is simple to design and synthesize, has low cost and high sensitivity and is accessible to a wide range of labels. This class of probes should find applications in a variety of areas wherever high specificity of nucleic acid hybridization is relevant.
The Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of Simple and reliable genotyping
technology is a key to success for high-throughput genetic screening
in the post-genome era. Here we have developed a new real-time PCR genotyping
approach that uses displacement hybridization-based probes:
displacing probes. The specificity of displacing probes could be simply assessed
through denaturation analysis before genotyping was implemented, and the probes
designed with maximal specificity also showed the greatest detection
sensitivity. The ease in design, the simple single-dye labeling chemistry and the
capability to adopt degenerated negative strands for point mutation genotyping make the
displacing probes both cost effective and easy to use. The feasibility of
this method was first tested by detecting the C282Y mutation in the human
hemochromatosis gene. The robustness of this approach was then validated by
simultaneous genotyping of five different types of mutation in the human
beta-globin gene. Sixty-two human genomic DNA samples with nine known genotypes were
accurately detected, 32 random clinical samples were successfully screened and 114
double-blind DNA samples were all correctly genotyped. The combined merits of
reliability, flexibility and simplicity should make this method suitable for
routine clinical testing and large-scale genetic screening.
http://www.molbiotech.chalmers.se/research/mk/lightup/lightup.htm
Vanvik et al.
developed light-up probes for sequence specific detection of nucleic
acids in homogeneous solution. The probes are made of the nucleic acid
analogue, PNA, and an assymmetric cyanine dye, which upon bind binding
to nucleic acids becomes intesively fluorescent. Under optimum
conditions the probe fluorescence increases 50-fold upon binding to
target DNA and the fluorescence can be observed by the naked eye.
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N. Svanvik, G. Westman, W. Dongyuan & M. Kubista.
Light-up Probes Thiazole Orange Conjugated PNA for Detection of Nucleic
Acid
in Homogeneous Solution. Anal. Biochem. 281, 26-35 (2000). |
| |
N. Svanvik, A. Stålberg, U. Sehlstedt, R. Sjöback
& M. Kubista. Detection of PCR Products in Real-time Using Light-up
Probes. Anal. Biochem. 287, 179-182 (2000). |
| |
N. Svanvik, J. Nygren, G. Westman & M. Kubista.
Free-probe Fluorescence of Light-up probes. J. Amer. Chem. Soc., 123,
803-809 (2001). |
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LUX primers have multiple advantages and among them:
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Faye Boeckman, Keith Hamby, and Larissa Tan, Bio-Rad
Laboratories,
2000
Alfred Nobel Drive, Hercules, CA 94547 USA
Yuanli Zhang1,2, Dabing Zhang2,*, Wenquan Li3, Jianqun Chen1, Yufa Peng4 and
Wei Cao3
Nucleic Acids Research, 2003, Vol. 31, No. 20 e123