Dyes & Fluorescence detection chemistry in qPCR
Navarro E, Serrano-Heras G, Castaño MJ, Solera J
Clin Chim Acta. 2015 439: 231-250
Real-time PCR is the method of choice in many laboratories for diagnostic and food applications. This technology merges the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction. Thus, the combination of excellent sensitivity and specificity, reproducible data, low contamination risk and reduced hand-on time, which make it a post-PCR analysis unnecessary, has made real-time PCR technology an appealing alternative to conventional PCR. The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far. Herein, different real-time PCR chemistries have been classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition, structures, mechanisms of action, advantages and applications of such real-time PCR probes and analogues are depicted in this review.
Randy Rasmussen Ph.D. (Idaho Technology, COO)
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. The biggest disadvantage of SYBR is that it binds to any dsDNA; the specific product, non-specific products and primer dimers are detected equally well. There are a number of ways to handle this problem. Careful optimization of the PCR reaction can usually reduce primer dimers to a level that is only important for very low copy detection. Hot start techniques like TaqStart antibody can be helpful in reducing primer dimer. The LightCycler Instrument allows melting curve analysis of the reaction. This can help to determine the fraction of the signal coming from the desired product and the fraction coming from primer dimer. Once the melting point of the product has been determined the LightCycler Instrument's flexible programming allows the user to acquire fluorescence above the melting temperature of the primer dimers, but below the melting temperature of the product.
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.
measurement at the end of the elongation step of every PCR cycle is
to monitor the increasing amount of amplified DNA. Together with a
melting curve analysis performed subsequently to the PCR, the SYBR
I format provides an excellent tool for specific product identification
Demonstration of preferential binding of SYBR
Green I to specific DNA fragments in real-time multiplex PCR
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, 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.
specific nucleic acid quantification and melting curve analysis.
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.
The most difficult qPCR applications demand double-quenched probes for optimum performance. BHQnova™ probes have improved quenching efficiency compared to traditional end-labeled probes, while also improving signal release upon amplification. BHQnova is most advantageous in longer probe designs, typically those over 25 bases, to boost the signal-to-noise ratio by overcoming the upper limit on sequence length.
For more product information, visit: our BHQnova Probes Product Info Page
The unique combination of online available assay design software and only 165 prevalidated, real-time PCR probes allows to quantify virtually any transcript in the transcriptomes of a large number of organisms. Universal ProbeLibrary probes are fully compatible with commonly used PCR conditions and the hydrolysis probe detection format. They are labeled at the 5' end with fluorescein (FAM) and at the 3' end with a dark quencher dye.
Flexibility, specificity, convenience - all in one with the Universal ProbeLibrary
The Universal ProbeLibrary combines the flexibility, availability and covenience of SYBR Green I assays with the specificity of Hydrolysis Probe assays. Just 165 prevalidated probes, that can easily be stored in your freezer are sufficient to quantify virtually any transcript from the transcriptomes of a large number of organisms. Target specific intron-spanning qPCR assays are designed online with the ProbeFinder software, freely available at the Universal ProbeLibrary Assay Design Center. The complete assay information, including the sequence of specific primer pairs, and the appropriate Universal ProbeLibrary probe, probe location, amplification product, is displayed on the result page.
more info here => Universal ProbeLibrary
Hybridization Probes for the Detection of Nucleic Acids in Homogeneous Solutions
Department of Molecular Genetics, Public Health Research Institute
225 Warren Street, Newark, NJ 07103, USA
Table of contents:
Wish Upon A Star: Molecular Beacons: Real Time in a Twinkle
While Saving Time: Intergen's Amplifluor allows
Direct Detection of PCR Products
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).
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
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.
Displacing probes - A new class of homogeneous nucleic acid probes based on specific displacement hybridization.
Li Q, Luan G, Guo Q, Liang J.
Nucleic Acids Res. 2002 Jan 15;30(2):E5.
The Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of
Education, Xiamen 361005, Fujian, Chinas
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.
Real-time PCR genotyping using displacing probes.
Cheng J, Zhang Y, Li Q.
Nucleic Acids Res. 2004 Apr 15;32(7):e61.
The Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of
Education, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian, China
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.
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.
| N. Svanvik, G. Westman, W. Dongyuan & M. Kubista.
Light-up Probes Thiazole Orange Conjugated PNA for Detection of Nucleic
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,
Measure the expression level of any human gene
the BD QZyme™ Assay for quantitative PCR (qPCR), a
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LUX (Light Upon
A brochure and
a manual is available on this product
to enable you to have a better idea of the capacity of our product.
LUX primers have multiple advantages and among them: