Enhancements and modifications of primer design program Primer3

Kõressaar, Triinu; Remm, Maido

doi:10.1093/bioinformatics/btm091

Cited by 2,284 publications

(1,645 citation statements)

References 15 publications

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“…CRISPR/Cas9‐generated single‐cell clones were isolated using DNeasy Blood and Tissue Kit (Qiagen). To detect mutations, primer pairs complementary to 100–150 base pairs up‐ and downstream of the sgRNA target region were designed using Primer3 database (Koressaar & Remm, 2007). The obtained primer sequences were fused to adapter sequences for the 2 nd PCR.…”

Section: Methodsmentioning

confidence: 99%

ERAD‐dependent control of the Wnt secretory factor Evi

et al. 2018

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Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of β‐catenin by the ubiquitin‐proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)‐associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP‐dependent manner. Ubiquitination of Evi involves the E2‐conjugating enzyme UBE2J2 and the E3‐ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD‐dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions.

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Section: Methodsmentioning

confidence: 99%

ERAD‐dependent control of the Wnt secretory factor Evi

et al. 2018

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“…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.…”

Section: Methodsmentioning

confidence: 99%

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

Kusche

Côté

Hernández

et al. 2017

Ecology and Evolution

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Age at maturity is a key life‐history trait of most organisms. In anadromous salmonid fishes such as Atlantic Salmon (Salmo salar), age at sexual maturity is associated with sea age, the number of years spent at sea before the spawning migration. For the first time, we investigated the presence of two nonsynonymous vgll3 polymorphisms in North American Atlantic Salmon populations that relate to sea age in European salmon and quantified the natural variation at these and two additional candidate SNPs from two other genes. A targeted resequencing assay was developed and 1,505 returning adult individuals of size‐inferred sea age and sex from four populations were genotyped. Across three of four populations sampled in Québec, Canada, the late‐maturing component (MSW) of the population of a given sex exhibited higher proportions of SNP genotypes 54Thrvgll3 and 323Lysvgll3 compared to early‐maturing fish (1SW), for example, 85% versus 53% of females from Trinité River carried 323Lysvgll3 (n MSW = 205 vs. n 1SW = 30; p < .001). However, the association between vgll3 polymorphism and sea age was more pronounced in females than in males in the rivers we studied. Logistic regression analysis of vgll3 SNP genotypes revealed increased probabilities of exhibiting higher sea age for 54Thrvgll3 and 323Lysvgll3 genotypes compared to alternative genotypes, depending on population and sex. Moreover, individuals carrying the heterozygous vgll3 SNP genotypes were more likely (>66%) to be female. In summary, two nonsynonymous vgll3 polymorphisms were confirmed in North American populations of Atlantic Salmon and our results suggest that variation at those loci correlates with sea age and sex. Our results also suggest that this correlation varies among populations. Future work would benefit from a more balanced sampling and from adding data on juvenile riverine life stages to contrast our data.

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“…Sanger sequencing primers were designed using the Primer 3 tool. 20,21 Target sequences were amplified using primers and conditions listed in Supplementary Methods. The BigDye Terminator system (Applied Biosystems) was used for sequencing on a 3730 DNA Analyzer (Applied Biosystems).…”

Section: Validation Of Variants By Sanger Sequencingmentioning

confidence: 99%

Breast ductal carcinoma in situ carry mutational driver events representative of invasive breast cancer

et al. 2017

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The spectrum of genomic alterations in ductal carcinoma in situ (DCIS) is relatively unexplored, but is likely to provide useful insights into its biology, its progression to invasive carcinoma and the risk of recurrence. DCIS (n = 20) with a range of phenotypes was assessed by massively parallel sequencing for mutations and copy number alterations and variants validated by Sanger sequencing. PIK3CA mutations were identified in 11/20 (55%), TP53 mutations in 6/20 (30%), and GATA3 mutations in 9/20 (45%). Screening an additional 91 cases for GATA3 mutations identified a final frequency of 27% (30/111), with a high proportion of missense variants (8/30). TP53 mutations were exclusive to high grade DCIS and more frequent in PR-negative tumors compared with PR-positive tumors (P = 0.037). TP53 mutant tumors also had a significantly higher fraction of the genome altered by copy number than wild-type tumors (P = 0.005), including a significant positive association with amplification or gain of ERBB2 (Po 0.05). The association between TP53 mutation and ERBB2 amplification was confirmed in a wider DCIS cohort using p53 immunohistochemistry as a surrogate marker for TP53 mutations (P = 0.03). RUNX1 mutations and MAP2K4 copy number loss were novel findings in DCIS. Frequent copy number alterations included gains on 1q, 8q, 17q, and 20q and losses on 8p, 11q, 16q, and 17p. Patterns of genomic alterations observed in DCIS were similar to those previously reported for invasive breast cancers, with all DCIS having at least one bona fide breast cancer driver event. However, an increase in GATA3 mutations and fewer copy number changes were noted in DCIS compared with invasive carcinomas. The role of such alterations as prognostic and predictive biomarkers in DCIS is an avenue for further investigation.

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Enhancements and modifications of primer design program Primer3

Cited by 2,284 publications

References 15 publications

ERAD‐dependent control of the Wnt secretory factor Evi

ERAD‐dependent control of the Wnt secretory factor Evi

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

Breast ductal carcinoma in situ carry mutational driver events representative of invasive breast cancer

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