Reporter groups on oligonucleotides

Real-time PCR Probes

The Real-time PCR or 5'-Nuclease assay is a sensitive assay for online monitoring of amplification kinetics. Real-time PCR Probes consist of an oligonucleotide complementary to the amplified probe. The probe is labeled at the 5'-end with the fluorescence donor (usually Fluorescein, HEX or TET) and a few bases downstream or on the 3'-end with a quencher, usually TAMRA. When no complementary sequence is available the fluorescence of the donor is quenched. During PCR the labeled oligonucleotide hybridizes to the target sequence and the 5'-dye is removed by 5'3'-exonuclease activity of Taq. The fluorescence of the donor is no longer quenched and can be measured. The fluorescence intensity is proportional to the amount of PCR-product formed during the early exponential phase of PCR (threshold value). In the probe design it is important that the melting point of the probe is higher than that of the upstream primer. The 3'-end of the probe must be protected against chain elongation during PCR. For blocking the 3'-end phosphate, cordycepin, 2',3'-dideoxynucleosides, inverseT or the quencher dye itself can be used. Labeling the 3'-end with TAMRA or another quencher can be achieved by using a 3'-amino-linker. Labeling internal positions of the sequence is possible by incorporating a modified thymidine residue which contains an amino-link at the 5-position of the base. This attachment does not interfere with hybridization. Figure & Reference

Principle of Real-time PCR

We offer

• a great variety of oligonucleotides with different labels and their combinations
• 5´- , 3´- or internal labels
• high purity probes and thorough quality control

New! Order information for frequently used Real-time PCR probes

Real-time PCR detection of 18sRNA with 5´ FAM, 3´ TAMRA probe. Amplification plots with (left to right) duplicate samples of 10, 1 and 0.1 ng RNA

Order online product amount cat. no. price/coupling Real-time PCR probe 5' FAM 3' TAMRA 5 nmol 5-0901-002 135.00 EUR 25 nmol 5-0901-003 175.00 EUR 100 nmol 5-0901-004 330.00 EUR Real-time PCR probe 5' HEX 3' TAMRA 5 nmol 5-0901-012 185.00 EUR 25 nmol 5-0901-013 255.00 EUR 100 nmol 5-0901-014 425.00 EUR Real-time PCR probe 5' TET 3' TAMRA 5 nmol 5-0901-022 139.00 EUR 25 nmol 5-0901-023 225.00 EUR 100 nmol 5-0901-024 355.00 EUR Order information see also "how to order" We offer a large selection of dye combinations suitable for Real-time PCR probes. Please request if your combination of interest is not on this list.