Scorpion Primer Services

Scorpion primers are a class of DNA single-stranded oligonucleotides established by Whitcombe et al. in 1999. It's generally believed that Scorpion primers adopt a unimolecular probing mechanism, which is different from other dual-labeled probes with biomolecular mechanisms such as Molecular Beacons and TaqMan probes. Together with their excellent properties, Scorpion probes can be used in multiple different fields, such as real-time PCR, endpoint PCR, SNP detection and gene quantification.

Structure and Working Mechanism

Figure 1 shows the detailed probing mechanism of a typical Scorpion primer. Like other dual-labeled probes, the common parts include a probe sequence held in a hairpin-loop structure and a self-complementary stem structure with a fluorophore at 5'-end quenched by a moiety in the vicinity of the 3'-end. Notably, the quencher is linked to a primer via a PCR blocker that can prevent read-through. The process could be divided into five steps: (a) initial denature by heating, when the primer is ready to bind target; (b) annealing of primer to target; (c) PCR extension from the primer; (d) the second cycle of denaturation and annealing, generating a new single-strand target molecule with the primer attached; and (e) the intramolecular binding between the loop region and the target, allowing fluorescence emitted.

Figure 1. Mechanism of Scorpion Primer DNA probe operation. (Thelwell N, et al., 2000)

Advantages

Based on its working mechanism, the generated undesired hybrids during the process are less stable than the reformed stem region. Besides, the blocker can efficiently reduce non-specific PCR products like primer dimer or mispriming events. Consequently, the signal can only be detected when the probe sequence within Scorpion primer perfectly hybridizes to target sequence. Scorpion primers are more efficient compared with Molecular Beacons and TaqMan probes and selective and sensitive enough for detecting single base mutations. Therefore, Scorpion primers can serve as a quite useful tool for biochemical detections.

Our Services

Creative Biogene can provide highly sensitive, sequence-specific Scorpion primers for multiple purposes, including SNP and pathogen detections, allele discrimination, multiplexing, viral load quantification, gene-related analysis and endpoint genotyping, etc.

All the bio-synthetic Scorpion primers are available, the corresponding parameters are listed below:

• About 100-200 bp of amplicon
• Structure test of designed primers will be performed; the proportion of secondary structure should be as low as possible compared with that of hairpin structure
• Tm_(stem) - Tm_(probe) > 5-10 ºC
• The lengths of probe, stem and corresponding target sequences should be approximately 17-27 bases, 6-7 bases and 11 bases, respectively;
• Most bases of stem sequence should be Cs and Gs and avoid motifs; C instead of G should place at the 5'-end in order to avoid quenching fluorophore
• QC will be provided, including MALDI-TOF MS and analytical HPLC