Over the last 15 years the polymerase chain reaction (PCR) has revolutionized the detection of Nucleic Acids and became an essential part of most laboratories involved in biomedical research by having proven to be a versatile tool in molecular biology. The use of this technique has generated unprecedented advances in gene discovery, diagnostics, and gene expression analysis because as little as a single copy of a particular sequence can be specifically amplified. Thus, attograms of nucleic acid which is far to little to be analyzed directly or used in biochemical reactions are quickly turned into as much as a microgram of DNA, which is more than enough for detection, sequencing or cloning. Theoretically, there is a quantitative relationship between amount of starting target sequence and amount of PCR product at any given cycle. However, by nature, an exponential amplification is not ideally suited to quantification as small differences in amplification efficiency between samples can become huge differences in results when they are multiplied as much as forty doublings. In practice, it is also a common experience for replicate reactions to yield different amounts of PCR products because in traditional PCR the amplification reaction continues until some reactants become limiting and the amount of product is therefore not directly related to the initial number of DNA copies in the sample.
Real-time/Quantitative (q)-PCR is a powerful advancement of the basic PCR technology. Through the use of appropriate fluorescent detection strategies in conjunction with proper instrumentation, the starting amount of nucleic acid in the reaction can be quantified. Quantification is achieved by measuring the increase in fluorescence during the exponential phase of PCR. Quantitative PCR has a sensitivity five orders of magnitude better than the best blotting procedures and a dynamic range of 10 orders of magnitude! These features made the work of turning PCR into a quantitative tool worthwhile which is reflected by the continuing increase of popularity for different kinds of real time/quantitative PCR Systems.
Applications of q-PCR are found in many key areas including:
- Gene Therapy Distribution and Expression Assays
- Drug Response Analysis
- Cell Bank Copy Number Analysis
- Mutation Analysis/SNP Analysis
- Expression System Development
- Residual DNA Analysis
- Detection of Pathogens
- GMO Analysis
- Human and Veterinary Diagnostics
- Blood products quality control
metabion can supply you probes for every real-time PCR instrument available in the market. No matter if you work with any of ABI's Prism 7XXX line machines, Corbett's Rotogene, BioRad's I-Cycler, Cepheid's Smart Cycler, MJ Research's Opticons, or Roche's LightCycler® - we have a solution for you.
For a list of compatible probes and equipment (.pdf)
Dual labelled fluorogenic probes
metabion's dual labelled fluorogenic probes are optimized for use in real-time quantitative PCR. As for this kind of oligonucleotides perfect quality to ensure reproducibility is a must, we have optimized our synthesis and purification protocols in a way that the background noise will be decreased to an absolute minimum. Offering BHQ dyes as a new class of dark quenchers that prevents fluorescence until a hybridization event occurs, and additionally do not display any native fluorescence, background problems seen with other quenchers are virtually eliminated. We will reliably guarantee top quality probes for your successful DNA/RNA detection experiments!
Our range of reporter-quencher combinations which suit Taqman® - as well as Molecular Beacon® chemistry should cover your needs for single or multiplex real-time PCR assays:
| Quenchers | ||||||||
|---|---|---|---|---|---|---|---|---|
| Reporters | TAMRA | BHQ-1 | BHQ-2 | BHQ-3 | Dabcyl | Abs (nm) | Em (nm) | Color |
| 6-FAM | Y | Y | N | N | Y | 495 | 520 | Yellow-Green |
| TET | Y | Y | N | N | Y | 521 | 536 | Yellow-Green |
| JOE | Y | Y | N | N | Y | 520 | 548 | Yellow |
| CAL Fluor 540 | N | Y | N | N | N | 522 | 541 | Yellow |
| Hex | Y | Y | Y | N | Y | 535 | 556 | Yellow |
| CAL Fluor 560 | N | Y | N | N | N | 537 | 558 | Yellow-Orange |
| TAMRA | N | N | Y | N | Y | 546 | 579 | Yellow-Orange |
| Cy3 | N | N | Y | N | N | 550 | 570 | Yellow-Orange |
| ROX | N | N | Y | N | N | 576 | 601 | Orange |
| CAL Fluor 610 | N | N | Y | N | N | 590 | 610 | Red |
| Cy5 | N | N | Y | Y | N | 643 | 667 | Red |
| Cy5.5 | N | N | N | Y | N | 675 | 694 | Red |
| IRD 700 | N | N | N | Y | N | 685 | 700 | Red |
| N = no, Y = yes | ||||||||
LightCycler® Probes
LightCycler® Probes from metabion are synthesized in our ISO-certified manufacturing facility using processes licensed by Roche. Unlike the TaqMan® assay where a self-quenching fluorogenic probe is cleaved to generate a fluorescent signal, the LightCycler® hybridisation probe system is based on two oligonucleotide probes that hybridize, in a head-to-tail arrangement, to adjacent sequences of the target DNA. Each probe carries a different marker dye, the first one 3'Fluorescein, the second one either 5'LC610, 5'LC640, 5`LC670 or 5'LC705.
| LightCycler® probes | |||
|---|---|---|---|
| Dye | Abs (nm) | Em (nm) | Color |
| LC Fluorescein | 495 | 520 | Yellow-Green |
| LC Red 610 | 590 | 610 | Red |
| LC Red 640 | 625 | 640 | Red |
| LC Red 670 | 650 | 670 | Red |
| LC Red 705 | 685 | 705 | Red |
Additionally, LC Probes' 3'OH terminus is "capped" by a phosphate group to prevent polymerase from extending the primer during PCR reaction. As the marker dyes are in close proximity, there can be a fluorescence resonance energy transfer (FRET) between them which allows a specific and quantitative real-time detection of the amplification of a nucleic acid template. To avoid sterical problems between the 3' Fluo and 5' LC Red end labels of the probes there should be a spacer of 1 to 5 nucleotides (4 to 25 Å distance) to separate the two probes from each other.
Click here for more information about our LightCycler® Probes
Useful link: www.gene-quantification.info
LightCycler® Probes are sold under license from HoffmannLa-Roche Ltd. No license under these patents to use the PCR Process is conveyed expressly or by implication to the purchaser by the purchase of this product. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Roche Molecular Systems Inc., 1145 Atlantic Avenue, Alameda, California 94501. Custom synthesis of LC Probes and oligonucleotides containing LC Red dyes under licence of Roche Diagnostics.
"TaqMan" and "LightCycler" are registered trademarks of Roche Molecular Systems Inc., "Prism" is a registered trademark of Applied Biosystems. "iCycler", "Opticon2" and "Chromo 4" are registered trademarks of Bio-Rad Laboratories. "SmartCycler" and "SmartCycler II" are registered trademarks of Cepheid. "Rotor-Gene" is a trademark of Corbett Research. "MX 3000P" and "MX4000" are registered trademarks of Stratagene, Inc.
All products offered for sale by metabion international AG are sold under the condition that they are used for research purposes only.