PRISE2: software for designing sequence-selective PCR primers and probes.

BackgroundPRISE2 is a new software tool for designing sequence-selective PCR primers and probes. To achieve high level of selectivity, PRISE2 allows the user to specify a collection of target sequences that the primers are supposed to amplify, as well as non-target sequences that should not be amplified. The program emphasizes primer selectivity on the 3' end, which is crucial for selective amplification of conserved sequences such as rRNA genes. In PRISE2, users can specify desired properties of primers, including length, GC content, and others. They can interactively manipulate the list of candidate primers, to choose primer pairs that are best suited for their needs. A similar process is used to add probes to selected primer pairs. More advanced features include, for example, the capability to define a custom mismatch penalty function. PRISE2 is equipped with a graphical, user-friendly interface, and it runs on Windows, Macintosh or Linux machines.ResultsPRISE2 has been tested on two very similar strains of the fungus Dactylella oviparasitica, and it was able to create highly selective primers and probes for each of them, demonstrating the ability to create useful sequence-selective assays.ConclusionsPRISE2 is a user-friendly, interactive software package that can be used to design high-quality selective primers for PCR experiments. In addition to choosing primers, users have an option to add a probe to any selected primer pair, enabling design of Taqman and other primer-probe based assays. PRISE2 can also be used to design probes for FISH and other hybridization-based assays

A noninvasive molecular approach: exploiting species-locus-specific PCR primers in defeating numts and DNA cross-contamination of cercopithecidae

The lack of a standardized, noninvasive molecular approach to studying genetic aspects of primates has made it hard for primatologists to decode the evolutionary history of these species. Researchers must optimize their own techniques to fully exploit the available samples. Lack of species-locus-specific primers also contributes to difficulties in using noninvasive genetic samples. Thus, the objectives of this study were to develop a standardized technique to collecting samples noninvasively, propose newly designed species-locus-specific primers, and optimize conditions for polymerase chain reaction (PCR) for Macaca fascicularis, M. nemestrina, Trachypithecus cristatus, and T. obscurus. Nine new species-locus-specific primers for three different loci of mitochondrial DNA, namely D-loop, cytochrome oxidase subunit I (COI), and cytochrome b, were successfully designed. These primers proved to be efficient in amplifying larger datasets (up to ~1,000 bp) of the targeted species in the optimized PCR conditions. The species-locus-specific primers are able to anneal to host DNA alone in highly contaminated feces of highlighted species. They can also offer alternatives measures in avoiding contamination related to nuclear insertion of mitochondrial pseudogenes (numts)

MCMC-ODPR : primer design optimization using Markov Chain Monte Carlo sampling

Background Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. Results After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. Conclusions MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base

High-throughput sequencing of 16S rRNA gene amplicons : effects of extraction procedure, primer length and annealing temperature

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers

White spot syndrome virus (WSSV) transmission risk through infected cooked shrimp products assessed by polymerase chain reaction (PCR) and bio-inoculation studies

The aim of the study was to evaluate the resistance of white spot syndrome virus (WSSV) in shrimps (Penaeus monodon) to the process of cooking. The cooking was carried out at 1000C six different durations 5, 10, 15, 20, 25 and 30 min. The presence of WSSV was tested by single step and nested polymerase chain reaction (PCR). In the single step PCR, the primers 1s5 & 1a16 and IK1 & IK2 were used. While in the nested PCR, primers IK1 &IK2 – IK3 & IK4 were used for the detection of WSSV. WSSV was detected in the single step PCR with the primers 1s5 and 1a16 and the nested PCR with the primers IK1 and IK2 – IK3 & IK4 from the cooked shrimp samples. The cooked shrimps, which gave positive results for WSSV by PCR, were further confirmed for the viability of WSSV by conducting the bio-inoculation studies. Mortality (100%) was observed within 123 h of intra-muscular post injection (P.I) into the live healthy WSSV-free shrimps (P. monodon). These results show that the WSSV survive the cooking process and even infected cooked shrimp products may pose a transmission risk for WSSV to the native shrimp farming systems

Ten polymorphic microsatellite primers in the tropical tree caimito, Chrysophyllum cainito (Sapotaceae).

UnlabelledPremise of the studyWe developed microsatellite primers for the tropical tree Chrysophyllum cainito (Sapotaceae) to determine the native range of the species, investigate the origin of cultivated populations, and examine the partitioning of genetic diversity in wild and cultivated populations. •Methods and resultsWe developed 10 polymorphic primers from C. cainito genomic DNA libraries enriched for di-, tri-, and tetranucleotide repeat motifs. The loci amplified were polymorphic in samples collected from Jamaica and Panama. The number of alleles per locus ranged from two to 10 and three to 12, while observed heterozygosities ranged from 0.074 to 0.704 and 0.407 to 0.852 in Jamaica and Panama, respectively. •ConclusionsThe microsatellite primers will be useful in future population genetic studies as well as those aimed at understanding the geographic origin(s) of wild and cultivated populations