SimpleGT Probes© – Sondas de qPCR com uma única marcação fluorescente para a Genotipagem de SNP

As SimpleGT Probes© são projetadas especificamente para se ligar a sequências que contêm SNPs, produzindo resultados de diagnóstico pela da análise de curvas de melting, de forma semelhante ao sistema LightCycler® HybProbe, mas com uma diferença essencial: as SimpleGT Probes© requerem apenas uma única sonda fluorescente em vez de duas.

O princípio é simples: quanto mais forte a hibridização, maior a temperatura de melting. Pelo contrário, mutações como os SNPs enfraquecem a estabilidade de ligação das SimpleGT Probes©, resultando numa mudança na curva de melting — tornando-as uma ferramenta poderosa e eficiente para a genotipagem de SNPs.

Especificações da SimpleGT Probe©:

• Sonda de qPCR marcada com uma fluoresceína única.
• Marcação fluorescente interna para detecção de sinal (aquisição a 530 nm).
• Extremidade 3′ fosforilada para evitar a elongação durante a PCR.
• Purificada por HPLC e verificada por mass check para garantir a mais alta qualidade.
• Comprimento recomendado da sonda: 20 a 30 nucleotídeos. Comprimento máximo: 40 nucleotídeos.

Características padrão da SimpleGT Probe©:

• Marcação fluorescente interna ligada a um nucleotídeo de base universal. A terminologia da metabion para essa modificação é FLNI.
• Fosfato na extremidade 3′ para bloquear a elongação pela polimerase na sonda.
• Sem modificação na extremidade 5′!

Oferecemos as SimpleGT Probes© em 7 escalas padrão diferentes (de 5 até 200 nmol), com quantidades definidas entregues.

Pedido do Primers Oligos

SimpleGT Probes© – Single-Labeled qPCR Probes for SNP Genotyping

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 primers@frilabo.pt. 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.

For your convenience, the SimpleGT Probes© Flyer is available for download.