1B) the probe element, which has a fluorophore attached at its 5′-end, is annealed to a complementary oligonucleotide bearing a quencher at the 3′-end. The probe sequence is held in a hairpin loop conformation by complementary stem sequences on the 5′ and 3′ sides of the probe, placing the fluorophore in the proximity of the quencher so that collisional quenching occurs. The mode of action of a stem–loop Scorpion is shown in Figure 1A. In the present study we focus on the two-oligonucleotide ‘duplex’ format. Previously, attention has been focused on the single-oligonucleotide ‘stem–loop’ Scorpion format ( 1, 2, 7). Unlike Taqman“™ probes, Scorpions do not depend upon enzymic cleavage and, therefore, rapid PCR cycling is possible ( 2). The intramolecular probing mechanism of Scorpions offers significant advantages over other genotyping systems such as Taqman“™ ( 4), molecular beacons ( 5) and hybridisation probes ( 6) that all rely on bimolecular probing. Under these conditions the mismatched probe re-associates with the quencher element to become non-fluorescent, whereas the hybridised wild-type probe is separated from the quencher element and is fluorescent. In this application the fluorescence is monitored above the T m of the mismatch probe–target duplex and below the T m of the fully complementary probe–target duplex. Scorpions technology can be used in allelic discrimination ( 1, 2) and is effective in SNP genotyping ( 3).
This would lead to displacement of the quencher and an increase in fluorescence, even in cases where a non-specific PCR product, such as a primer dimer, is formed. The PCR stopper prevents undesirable read-through of the probe by Taq DNA polymerase.
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On cooling, the probe is free to bind to this complementary sequence, producing an increase in fluorescence, as the quencher is no longer in the vicinity of the fluorophore. After PCR extension of the Scorpion primer, the resultant amplicon contains a sequence that is complementary to the probe, which is rendered single-stranded during the denaturation stage of each PCR cycle. The basic elements of Scorpions in all formats are: (i) a PCR primer (ii) a PCR stopper to prevent PCR read-through of the probe element (iii) a specific probe sequence and (iv) a fluorescence detection system containing at least one fluorophore and quencher. Scorpion primers are new diagnostic tools for the specific detection of PCR products in real-time ( 1, 2). This research provided insight into genetic structuring of stranded porpoises in 2007, but data from multiple years should be included to be able to help estimate population sizes.Received JRevised JAccepted August 15, 2001. However, this was not corroborated by other indices of inbreeding. In contrast, heterozygosity levels were low, indicating some level of inbreeding in this population. The results of this study indicate that the Dutch population is panmictic. Furthermore, microsatellite analysis revealed no genotypic differences between seasons, locations or genders. However, microsatellite analysis showed that these mtDNA haplotypes did not represent separate groups on a nuclear level. mtDNA haplotypes were not randomly distributed along the Dutch coastline. mtDNA analysis showed 6 variable positions in the control region, defining 3 different haplotypes. We made use of both mitochondrial (mtDNA) and microsatellite DNA analysis of samples from dead stranded porpoises along the Dutch coast during 2007. We also tested whether there is a link between stranding and necropsy data, and genetic diversity. In this study we investigated genetic structure among animals stranded at different locations and times of year. In the Dutch part of the North Sea, sighting and stranding data are used to estimate population sizes, but these data give little insight into genetic structuring of the population.
Conservation management in the North Sea is often motivated by the population size of marine mammals, like harbor porpoises Phocoena phocoena.
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