Primer Design for Pyrosequencing v1

This is a protocol for the design of reliable primers for amplification of bisulfite-converted DNA with the intent of being used for pyrosequencing. It also includes a protocol for the design of sequencing primers for pyrosequencing and recommendations for optimization.

Primer design of D-loop region for wild population genetics of Rusa timorensis in Indonesia

Rusa timorensis (Javan deer) is endemic wildlife in Indonesia and is estimated at less than 10.000 individuals with continuously declining populations due to habitat loss and illegal hunting in the wild. This declining low population indicates a greater risk of extinction. Unfortunately, the genetic information of the wild Javan deer population for conservation management strategies still lacks data due to challenging sampling in the wild. Most recent studies were analysing the breeding populations outside Indonesia. Here, we propose the primer design of the D-loop genetic marker to determine the genetic population of wild Javan deer. We used metadata analysis of genetic sequences and new samples from five wild populations to design the specific primer of the D-loop region of the wild Javan deer in Indonesia. We used software, i.e.., Primer3 to design the primers, BLAST for specificity and Oligo Analyzer™ Tool for efficiency of the primer. The Annealing temperature optimisation started with pre-denaturation at 94 °C followed by 35 cycles of denaturation at 95°C; 51-56°C annealing for each one degree’s different per PCR treatment; and 72°C extensions. We successfully designed a specific primer (RL-3.1a) to amplify 235 bp of the D-loop region at 52°C annealing’s temperature.

The web-based multiplex PCR primer design software Ultiplex and the associated experimental workflow: up to 100- plex multiplicity

Background A large number of variants have been employed in various medical applications, such as providing medication instructions, disease susceptibility testing, paternity testing, and tumour diagnosis. A high multiplicity PCR will outperform other technologies because of its lower cost, reaction time and sample consumption. To conduct a multiplex PCR with higher than 100 plex multiplicity, primers need to be carefully designed to avoid the formation of secondary structures and nonspecific amplification between primers, templates and products. Thus, a user-friendly, highly automated and highly user-defined web-based multiplex PCR primer design software is needed to minimize the work of primer design and experimental verification. Results Ultiplex was developed as a free online multiplex primer design tool with a user-friendly web-based interface (http://ultiplex.igenebook.cn). To evaluate the performance of Ultiplex, 294 out of 295 (99.7%) target primers were successfully designed. A total of 275 targets produced qualified primers after primer filtration, and 271 of those targets were successfully clustered into one compatible PCR group and could be covered by 108 primers. The designed primer group stably detected the rs28934573(C > T) mutation at lower than a 0.25% mutation rate in a series of samples with different ratios of HCT-15 and HaCaT cell line DNA. Conclusion Ultiplex is a web-based multiplex PCR primer tool that has several functions, including batch design and compatibility checking for the exclusion of mutual secondary structures and mutual false alignments across the whole genome. It offers flexible arguments for users to define their own references, primer Tm values, product lengths, plex numbers and tag oligos. With its user-friendly reports and web-based interface, Ultiplex will provide assistance for biological applications and research involving genomic variants.

MetaFunPrimer: an Environment-Specific, High-Throughput Primer Design Tool for Improved Quantification of Target Genes

Amplification-based gene characterization allows for sensitive and specific quantification of functional genes. There is often a large diversity of genes represented for functional gene groups, and multiple primers may be necessary to target associated genes.