Editing TREC ASSAY (section)

== Probe Design ==

IDT has developed an internal ZEN quencher for the production of double-quenched probes that produce qPCR data with less background and increased signal. Doublequenched probes contain a 5’ fl uorophore choice of FAM, HEX, JOE, TET, or MAX; a 3’ IBFQ quencher; and an internal ZEN quencher. While the distance between fl uorophore and quencher in traditional probes is 20−30 bases, the internal ZEN quencher decreases this distance to only 9 bases . This shortened distance, particularly when combined with the traditional 3’ end quencher, leads to extremely effi cient quenching with signifi cantly less background, and enables the use of much longer probes for designs falling within AT-rich target regions. In addition to the greatly decreased background, the double-quenched probes allow users to experience increased sensitivity and greater precision in their qPCR experiments.

'''Tm''': The melting temperature of the probe should be 6−8°C higher than the primers and should fall within the range of 66−70°C for a standard two-step protocol. If the melting temperature is too low, the probe will not bind to the target. In this case, the primers may amplify a product, but as the probe is not bound to a target, it will not be proportionally degraded and, thus, will be unable to provide the fl uorescence that is necessary to detect the product.&nbsp;

'''Length''': The length of a single-quenched probe should be 20−30 bases to achieve an ideal Tm without increasing the distance between the dye and quencher such that the quencher will no longer absorb the fl uorescence of the dye (probes with a single terminal quencher that are longer than 30 bases may perform poorly due to the distance between the quencher and dye). Double-quenched probes that include the IDT ZEN molecule as a secondary, internal quencher allow longer probes to be used while providing strong quenching and increased signal. See Section 4.1.3 for more information.&nbsp;

'''GC content''': Aim for a GC content of 35−65% and avoid a G at the 5’ end to prevent quenching of the 5’ fl uorophore. As with the primers, avoid sequences that may create secondary structures or dimers.

'''Location''': Ideally, the probe should be in close proximity to the forward or reverse primer, but not overlap, although this is not absolutely necessary. Probes can be designed to bind to either strand of the target.

'''Amplicons Length''': Design amplicons of 70−150 bp, which will allow the primers and probe to compete for hybridization and provide a sequence that is long enough for all components to bind. This length is most easily amplified using standard cycling conditions. Longer amplicons of up to 500 bases can be generated, but cycling conditions will need to be altered to account for the increased extension time. Amplicons or assay designs should span an exon–exon junction to reduce the possibility of genomic contamination.&nbsp;

'''Tm''': Calculate all melting temperatures under real-time PCR conditions—standard parameters for qPCR are 50 mM K+, 3 mM Mg2+, and 0.8 mM dNTPs: however they can vary widely from this, particularly with respect to Mg2+ concentration. See Section 4.2.2d, below, for more information on calculating melting temperature.