metabion SimpleGT Probes© are the latest addition to our qPCR probes portfolio, producing diagnostic results by melting curve analysis similar to the LightCycler® HybProbe system.
However, while the LC® HybProbe concept requires two fluorescently labeled probes hybridizing side-by-side to the amplified target sequence and producing a FRET signal, SimpleGT Probes© assays need only one single fluorescent probe.
If a SimpleGT Probe© is free in solution, emission of the fluorescent dye is quenched by a naturally occurring nucleotide the dye is attached to (A). Upon probe hybridization to its target, quenching is reduced and reporter dye fluorescence emission increases (B). As a result, once hybridized to its target sequence, the SimpleGT Probe© emits more fluorescence than it does unhybridized. Changes in fluorescence are solely based on the hybridization status of the probe.
SimpleGT Probes© are designed to specifically hybridize to a target sequence that contains a SNP. The more stable hybridization between the SimpleGT Probe© and its target sequence, the higher the melting temperature of the formed duplex. Mutations such as SNPs weaken the stability of SimpleGT Probe© binding.
Key Features:
- Single-Labeled qPCR Probe Format - SLP Format as opposed to DLP Format.
- Single Probe System as opposed to HybProbes FRET system.
- Hybridization dependent melting curve analysis as opposed to endpoint analysis (Hybridization vs. Hydrolysis probes).
- Designed for applications on LightCycler® Platform, and applicable to a broad range of tech platforms performing melting curve analysis with similar parameters and settings like signal detection/acquisition at 530 nm.
Main Application Areas:
- Research: Provides an efficient solution for SNP genotyping and mutation detection in academic studies on human, animal, plant, environmental samples (water, air, soil).
- Clinical Diagnostics: Offers a precise and cost-effective mutation detection method for diagnosing and monitoring disease, providing prognoses, and predicting treatment outcome. Supports the development of genetic assays with customization options and license-free use.
Our Standard Portfolio
SimpleGT Probe© specs
- Single Fluorescein labeled qPCR probe.
- Internal Fluo label for signal detection (acquisition at 530 nm).
- Phosphorylated 3' end to avoid elongation during PCR.
- HPLC purified and Mass Check verified to ensure highest quality.
- Recommended probe length 20 - 30 nts. Maximum length 40 nts.
SimpleGT Probe© features by default
- Internal Fluo label attached to a universal base nucleotide. metabion proprietary terminology for this modification is FLNI.
- 3´ Phos for blocking Polymerase elongation of the probe.
- No 5´modification!
Standard scales – delivered quantity
Like for our LC® Probes we offer SimpleGT Probes© in 7 different standard scales at defined quantities delivered.
- 5 nmol
- 10 nmol
- 20 nmol
- 30 nmol
- 50 nmol
- 100 nmol
- 200 nmol
For larger amounts, large volume orders or other specials, please do not hesitate to contact us at info@metabion.com. Our team will be happy to advise you of feasibility and prices.
SimpleGT Probe© design considerations
- Don´t place the probe close to the 5´primer-binding site on the target strand.
- Probe´s Tm should be 5-10°C higher than primers´ Tms.
- The sequence covering the polymorphism should be no less than 3 nucleotides away from the probe´s 3´/5´ termini.
- Internal positioning of the Fluorescein label is mandatory, as signal dynamics have a great advantage over terminal labeling.
- Place the internal label no less than 3 nts from either end (3´/5´) of the probe.
- Don´t place the label too close to the polymorphic site, but distance it no less than 2 nts from the SNP (up- and downstream).
- Avoid reverse complementary dG nts in the reverse complementary target strand in -1, 0, and +1 position relative to the internal label.
- Note that the internal fluorescent dye label is attached to a universal base deoxyribosyl-derivative. The universal base pairs equally with all four naturally occurring DNA/RNA bases, retaining structure and function of the probe-target sequence duplex. However, the probe´s Tm value will slightly decrease by less than 3%.
- 3´ Phosphorylation is mandatory to block probe elongation during PCR.
Order here or contact us at info@metabion.com for more information.
For your convenience, the SimpleGT Probes© Flyer is available for download in our Downloads section.