Dual-Labeled DNA Probes

Fluorescent Probes1

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To achieve accurate and reliable results from your real-time quantitative PCR, choose from the broadest range of sequence–specific, fluorescent probes:

All of our fluorescent probes are deprotected, desalted and purified by PAGE or RP-HPLC.

Every fluorescent probe is systematically quality-controlled by PAGE or mass spectrometry analysis.

What is real-time quantitative PCR?
Real-time quantitative PCR is an innovative and robust technique for quantitative analysis of gene expression, mutation detection, and SNP genotyping. Real-time quantitative PCR was developed to overcome the basic weakness of classical PCR technology: whereas PCR can indicate the presence of a particular nucleic acid in a sample after a completed PCR amplification, it cannot directly quantify the amount of amplicon.

Real-time quantitative PCR systems quantify the amount of amplified product through the detection and quantitation of a fluorescent dye. This dye, upon excitation, emits a light or signal that increases in intensity in direct proportion to the amount of amplified PCR product and, thereby, quantifies the amount of target DNA. The assays are directly performed in a single, closed tube without any purification or separation step, avoiding risks of contamination and the need for post-PCR handling.

Real-time quantitative PCR systems also enable mutation detection via melting-curve analysis, as each double-stranded DNA product has its own specific melting temperature (Tm). Melting-curve data enables the researcher to differentiate between specific PCR products and non-specific PCR products, such as primer-dimers.

Why use sequence-specific probes?
Detection by real-time quantitative PCR can be sequence-specific or non sequence specific, and the method chosen will effect the performance of your real-time quantitative PCR results. For instance, SYBR® Green 1, a non-sequence-specific reagent, can bind to any double-stranded DNA including primer-dimers and other non specific reaction products, and so may result in an over-estimation of the amount of target DNA. However, sequence-specific probes bind only to the target template and therefore achieve accurate and reliable quantification of the amount of target DNA.

Specifications of Fluorescent Probes from Sigma Custom Products for Real-Time Quantitative PCR

Quality control

All of our oligonucleotides undergo vigorous process monitoring and strict quality control. Length and labeling are systematically controlled by PAGE or mass spectrometry analysis. Quantity is measured by UV Absorbance at 260 nm.

Fluorescent probes are systematically quality-controlled by RP-HPLC. Additionally, the ratio between dye absorbance (nm) and DNA/RNA absorbance (nm) is calculated for the main product peak. This ratio defines the purity of the labeled probe, demonstrating that no contamination from partially labeled or unlabeled probes exists.

Purification

Fully deprotected and desalted. Purified by PAGE or RP-HPLC

Length

LightCycler probes:

15 to 40 mers (optimal length: 20 to 30 mers)

Dual-labeled fluorogenic probes:

15 to 40 mers

Molecular Beacons:

15 to 40 mers

Scorpions probes:

30 to 60 mers (uni-probe)
15 to 45 mers (bi-probe)

Bases

DNA (A, C, G or T)

Backbone

Phosphodiester bond

Format

Delivered as dried-down product in opaque tubes

Turn-around time (TAT)

LightCycler probes:

4 to 5 working days

Dual-labeled fluorogenic probes:

5 to 6 working days

Molecular Beacons:

5 to 6 working days

Scorpions probes:

7 to 10 working days

Turn-around time is dependent upon successful QC validation and does not include shipping time.

Storage and stability

Although oligonucleotides are stable in solution at 4°C for up to 2 weeks, Sigma Custom Products recommends storage at –20°C. Repetitive freeze-thaw cycles should be avoided by storing as aliquots. Storing at concentrations above 20 µM is recommended. Oligonucleotides with fluorescent labels should be protected from exposure to light and air. Sigma Custom Products guarantees its oligonucleotides for six months, when stored under the above conditions.

Shipment

Shipped by mail or express delivery, dry, in individual, opaque tubes

Oligonucleotide
Technical Data
Sheet

Oligonucleotides are delivered with an OligonucleotideTechnical Data Sheet, which includes oligonucleotide name, sequence, concentration, precise quantity in OD and nmols, Tm, MW, length, extinction coefficient and purification data.

Analysis Certificate

An HPLC analysis certificate is also supplied in addition to the Oligonucleotide Technical Data Sheet.

Services available upon request

• Probe design services
• High-throughput screening formats (microplates, etc)
• Aliquoting
Additional services may increase turn-around time

Pricing

Please contact your local Sigma Custom Products representative

Ordering

On-line,by email or fax


Design Service for Fluorescent Probes
Save time by letting Sigma Custom Products design your fluorescent probes and associated primers for your real-time quantitative PCR applications or mutation detection needs.

Using our in-house expertise and powerful design tools, we will provide you with the optimal probe design. Should you work with SYBR Green I and need primer sequences, we recommend that we also design the appropriate fluorescent probe at the same time.

Our team of design specialists is always available to answer any questions you may have about our design service. Please contact us at oligo.design@proligo.com.

Note: This design service applies only to probes and primers for real-time quantitative PCR applications and mutation detection needs. If you have already designed your own primer and/or probe sequence, we can also test your design.