Primer3—new capabilities and interfaces

Abstract

Polymerase chain reaction (PCR) is a basic molecular biology technique with a multiplicity of uses, including deoxyribonucleic acid cloning and sequencing, functional analysis of genes, diagnosis of diseases, genotyping and discovery of genetic variants. Reliable primer design is crucial for successful PCR, and for over a decade, the open-source Primer3 software has been widely used for primer design, often in high-throughput genomics applications. It has also been incorporated into numerous publicly available software packages and web services. During this period, we have greatly expanded Primer3’s functionality. In this article, we describe Primer3’s current capabilities, emphasizing recent improvements. The most notable enhancements incorporate more accurate thermodynamic models in the primer design process, both to improve melting temperature prediction and to reduce the likelihood that primers will form hairpins or dimers. Additional enhancements include more precise control of primer placement—a change motivated partly by opportunities to use whole-genome sequences to improve primer specificity. We also added features to increase ease of use, including the ability to save and re-use parameter settings and the ability to require that individual primers not be used in more than one primer pair. We have made the core code more modular and provided cleaner programming interfaces to further ease integration with other software. These improvements position Primer3 for continued use with genome-scale data in the decade ahead.

Characterization of natural variation in North American Atlantic Salmon populations (Salmonidae: Salmo salar) at a locus with a major effect on sea age

“…Library preparation followed a two‐step PCR design, with PCR1 targeting the candidate region and attaching the Illumina adaptors and PCR2 adding traceable barcodes for each individual to the amplicons. The PCR1 primers targeting the SNPs causing nonsynonymous amino acid changes in the vgll3 protein (Primer set “VGLL3_54” targeting Met54Thr vgll3 and “VGLL3_323” targeting Asn323Lys vgll3 ) and the akap11 protein (“AKAP11” targeting Val214Met akap11 ) , as well as for the SIX6 TOP SNP (“SIX6”), were designed using Primer3 (Koressaar & Remm, 2007; Untergasser et al., 2012) based on the information provided in recently published manuscripts (Ayllon et al., 2015; Barson et al., 2015) and the nucleotide and amino acid sequences for vgll3 , akap11, and six6 of the Atlantic Salmon reference genome deposited at GenBank (GCA_000233375.4) (Lien et al., 2016) (Table S2). Target lengths ranged between 406 and 444 bp.…”

De novo assembly and characterization of the Hucho taimen transcriptome

“…For SSRs, we used modified Sputnik (La Rota, Kantety, Yu, & Sorrells, 2005) to search di‐, tri‐, tetra‐, and pena‐nucleotide unit SSRs, and Primer3 (Untergasser et al., 2012) was then used to design the primers. For the SNPs, we first use Bowtie2 (Langmead & Salzberg, 2012) for mapping clean reads to transcripts and then called SNPs using SAMtools (Li et al., 2009).…”

Genetic alterations of differentiated thyroid carcinoma in iodine‐rich and iodine‐deficient countries

“…PCR amplified exons 2 and 3 of each RAS gene followed by Big Dye terminator cycle‐sequencing reaction and sequence reading on an ABI PRISM 3130 genetic analyzer (Applied Biosystems, Foster City, CA). All primers for NRAS, HRAS, and KRAS exons 2 and 3 were designed with Primer3 software 34. Table 1 provides primer details.…”