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 beeing far too 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 the amount of starting target sequence and the 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 during 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 a seasoned selection of Quenchers trimmed to cover our Reporter portfolio´s emission spectra, we will reliably guarantee top quality probes for your successful DNA/RNA detection experiments!
Our range of reporter-quencher combinations which suit probe hydrolysis as well as probe hybridisation based assays should cover your needs for single or multiplex real-time PCR applications.
Honoring our customers´ demand for seizable delivery amounts based on final yields in nmoles rather than abstract synthesis scales indicating to be expected OD260 values, we are introducing a transparent and best price-performance scheme for our broad selection of dual labeled fluorogenic probes.
Pointing out some key aspects:
- 5 scales based on delivered quantities tailored to your routine needs (see table 3).
- Length independent guaranteed (range) of delivered quantities in nmoles for probes from 18 to 40mers. Please note that OD260 values are a measure of total nucleotides´ optical density. Hence, neither purity nor amount of ordered substance are transparently reflected. For simplification and exemplification reasons look at the following:
1 OD of the 20mer 5´CAT CGT ATT CGA TGC TAC GT 3´
translates into approximately 5 nmol.
1 OD of the 40mer 5´CAT CGT ATT CGA TGC TAC GT CAT CGT ATT CGA TGC TAC GT 3´
translates into approximately 2.5 nmol.
Therefore, a 1 OD guaranteed amount of delivered product can vary significantly , while metabion´s commitment to delivered yields in nmol for probes ranging from 18 to 40 nucleotides does not allow for ambiguity in terms of what you expect and pay for.
- metabion´s routine dual labeled probe portfolio perfectly covers the spectrum of applicable excitation and detection wavelengths. Reporter-Quencher combinations – 5´- 3´ (see table 6) and 3´- 5´ (see table 7) - are suitable for all commonly used Real Time PCR platforms. However, don´t hesitate to ask for non-listed combinations including internally or multiply labeled oligonucleotides.
- Comprehensive and best value pricing scheme (see table 8), which includes HPLC purification as well as Mass-Check QC for our high-quality products.
It matters what you actually get for your money!
Table 1: Reporter/3'Quencher combinations
| 3' Quencher | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| TAMRA | BHQ-1 | BHQ-2 | BHQ-3 | Eclipse | Dabcyl | ||||
| Abs range (nm) | 470-560 | 480-580 | 550-650 | 620-730 | 390-625 | 380-550 | |||
| Abs max (nm) | 544 | 535 | 579 | 672 | 522 | 453 | |||
| 5' Reporter | Pricing Level | Abs (nm)* | Em (nm)* | Color | |||||
| LC® CYAN500 | - | 3 | - | - | 3 | 3 | 450 | 500 | |
| Fluo | 1 | 1 | 1 | - | 1 | 1 | 495 | 520 | |
| 6-FAM | 1 | 1 | 1 | - | 1 | 1 | 495 | 520 | |
| Rhodamine Green | - | 3 | - | - | 3 | 3 | 503 | 528 | |
| TET | 2 | 2 | 2 | - | 2 | 2 | 521 | 536 | |
| JOE | 2 | 2 | - | - | 2 | 2 | 522 | 548 | |
| Yakima Yellow | 2 | 2 | - | - | 2 | 2 | 530 | 549 | |
| Hex | 1 | 1 | 1 | - | 1 | 1 | 535 | 556 | |
| Cy3 | - | - | 3 | - | 3 | - | 546 | 563 | |
| TAMRA | - | - | 3 | - | 3 | 3 | 564 | 579 | |
| ROX | 3 | - | 3 | - | 3 | - | 576 | 601 | |
| Texas Red | - | - | 3 | - | 3 | - | 586 | 610 | |
| LC®Red 610 | - | - | 3 | - | 3 | - | 590 | 610 | |
| LC®Red 640 | - | - | 3 | 3 | - | - | 625 | 640 | |
| Cy5 | - | - | 3 | 3 | - | - | 646 | 662 | |
| Cy5.5 | 3 | - | 3 | 3 | - | - | 683 | 705 | |
| IRD 700 | 3 | - | 3 | 3 | - | - | 685 | 705 | |
| *Please note that absorption and emission wavelengths are influenced by the solvent (pH, salt conditions, etc.), oligo sequence and other factors. | |||||||||
Table 2: 5´Quencher/3´Reporter combinations
| 3' Reporter | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Fluo | 6-FAM | JOE | HEX | CY3 | ROX | LC Red610 |
LC Red640 |
CY5 | CY5.5 | |
| Abs (nm)* | 495 | 495 | 522 | 535 | 556 | 576 | 590 | 625 | 646 | 683 |
| Em (nm)* | 520 | 520 | 548 | 556 | 563 | 610 | 610 | 640 | 662 | 705 |
| 5' Quencher | Pricing Level | |||||||||
| BHQ-1 | 2 | 2 | 3 | 3 | - | - | - | - | - | - |
| BHQ-2 | 2 | 2 | - | 3 | 3 | 3 | 3 | 3 | 3 | 2 |
| Eclipse | 1 | 1 | 2 | 3 | - | 3 | 3 | - | - | - |
| Dabcyl | 1 | 1 | 3 | - | - | - | - | - | - | - |
| *Please note that absorption and emission wavelengths are influenced by the solvent (pH, salt conditions, etc.), oligo sequence and other factors. | ||||||||||
Table 3: Dual labeled fluorogenic probes pricing
For internal Reporter/Quencher positioning please inquire. We will be happy to come back with our advise upon our feasibility check!
LightCycler® Probes
LightCycler® Probes from metabion are synthesized in our ISO-certified manufacturing facility using processes licensed by Roche. Unlike 5´nuclease chemistry based assays where a self-quenching fluorogenic probe is cleaved to generate a fluorescent signal (hydrolysis assays), 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 (hybridisation assays). Each probe carries a different marker dye, the first one 3'Fluorescein, the second one either 5'LC®610, 5'LC®640, 5`LC®670 or 5'LC®705.
Table 4: LC® Fluorophores
| LightCycler® probes - Fluorophores | |||
|---|---|---|---|
| Dye | Abs (nm) | Em (nm) | Color |
| LC® CYAN 500 | 450 | 500 | |
| LC® Fluorescein | 495 | 520 | |
| LC® Red 610 | 590 | 610 | |
| LC® Red 640 | 625 | 640 | |
| LC® Red 670 | 650 | 670 | |
| LC® Red 705 | 685 | 705 | |
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, fluorescence resonance energy transfer (FRET) between them takes place 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.
We are offering LC®probes in 7 different scales at defined molar amounts.
Table 5: LC® Probes - Scales
| LightCycler® Probes (single tube format) | |
|---|---|
| Delivered quantity (nmol)* | max. Length |
| 1 | 40mer |
| 2 | 40mer |
| 3 | 40mer |
| 5 | 40mer |
| 10 | 40mer |
| 20 | 40mer |
| 30 | 40mer |
| For larger quantities, please inquire | |
| * including HPLC and Mass Check | |
Within the range of applicable and feasible designs of LC® probes (Hyb-Probes) we are able to provide you with an exhaustive portfolio for utmost flexibility. Below tables shall give you a comprehensive layout of possible Hyb-Probe designs including pricing levels /pricing schedules respecting scales of delivery and degree of production difficulty.
Table 6: LC® Probes – 5´-3´label combinations and pricing levels
| 5´-3´label combinations | |||||||
|---|---|---|---|---|---|---|---|
| 3' label | |||||||
| 5' label | Phos | LC610 | LC640 | LC670 | LC705 | LC Fluo | unmodified |
| Pricing Level | |||||||
| LC® Cyan 500 | 4 | - | - | - | - | 3 | 2 |
| LC® Fluo | 3 | - | - | - | - | 3 | 1 |
| LC® Red 610 | 2 | 4 | - | - | - | 4 | 1 |
| LC® Red 640 | 2 | / | 5 | - | - | 4 | 1 |
| LC® Red 670 | 2 | 4 | 4 | 4 | - | 4 | 1 |
| LC® Red 705 | 2 | 5 | 5 | 5 | - | 4 | 1 |
| Phos | - | 3 | 3 | 3 | 4 | 2 | - |
| unmodified | 1 | 2 | 2 | 3 | 3 | 1 | - |
Table 7: LC® Probes – Internal-3´label combinations and pricing levels
| Internal-3´label combinations | |||||||
|---|---|---|---|---|---|---|---|
| 3' label | |||||||
| Internal label | Phos | LC610 | LC640 | LC670 | LC705 | LC Fluo | unmodified |
| Pricing Level | |||||||
| LC® Cyan 500 | - | - | - | - | - | - | - |
| LC® Fluo | 3 | - | - | - | - | 4 | 2 |
| LC® Red 610 | 4 | 5 | - | - | - | 5 | 4 |
| LC® Red 640 | 4 | - | 5 | - | - | 5 | 4 |
| LC® Red 670 | 5 | - | - | 5 | - | 5 | 3 |
| LC® Red 705 | 5 | - | - | - | 5 | 5 | 4 |
| Phos | - | - | - | - | - | - | - |
| unmodified | 1 | 2 | 2 | 3 | 3 | 1 | - |
Table 8: LC® Probes – 5´-Internal label combinations and pricing levels
| Internal-5´label combinations | |||||||
|---|---|---|---|---|---|---|---|
| Internal label | |||||||
| 5' label | Phos | LC610 | LC640 | LC670 | LC705 | LC Fluo | unmodified |
| Pricing Level | |||||||
| LC® Cyan 500 | - | - | - | - | - | 5 | 2 |
| LC® Fluo | - | - | - | - | - | 5 | 1 |
| LC® Red 610 | - | 5 | - | - | - | - | 1 |
| LC® Red 640 | - | - | 5 | - | - | - | 1 |
| LC® Red 670 | - | - | - | 5 | - | 5 | 1 |
| LC® Red 705 | - | 5 | 5 | 5 | 5 | 5 | 1 |
| Phos | - | 3 | 3 | 3 | 4 | 2 | - |
| unmodified | - | 3 | 3 | 3 | 4 | 2 | - |
Table 9: LC® probes** – Pricing
| LC® probes** – Pricing | ||||||
|---|---|---|---|---|---|---|
| Scales in defined molar amounts |
For probes </= 33-40 bases; additional base charge of € 4.00 for probes >33 nt Prices include HPLC purification and Mass-Check QC for highest purity and best quality! |
|||||
| Quantity (nmol) |
No. of tests** | 1 Price in € |
2 Price in € |
3 Price in € |
4 Price in € |
5 Price in € |
| 1 | 300 | 80,00 | 195,00 | 250,00 | 320,00 | 400,00 |
| 2 | 600 | 87,00 | 220,00 | 280,00 | 360,00 | 450,00 |
| 3 | 1000 | 95,00 | 250,00 | 350,00 | 450,00 | 565,00 |
| 5 | 1500 | 115,00 | 375,00 | 475,00 | 605,00 | 760,00 |
| 10 | 3000 | 250,00 | 490,00 | 590,00 | 755,00 | 945,00 |
| 20 | 6000 | 310,00 | 790,00 | 990,00 | 1270,00 | 1590,00 |
| 30 | 10000 | 375,00 | 920,00 | 1390,00 | 1780,00 | 2225,00 |
| For probe design, larger molar amounts, large volume orders or other specials, please inquire at info@mymetabion.com. | ||||||
* Values calculated for 3 pmol of probe per assay (range of optimal amount 2-10 pmol).
** 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 LCRed dyes under licence of Roche Diagnostics. All products are for research only. LightCycler® is a trademark of Idaho Technology Inc.
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.