Highly Parallel and Short-Acting Amplification with Locus-Specific Primers to Detect Single Nucleotide Polymorphisms by the DigiTag2 Assay

The DigiTag2 assay enables analysis of a set of 96 SNPs using Kapa 2GFast HotStart DNA polymerase with a new protocol that has a total running time of about 7 hours, which is 6 hours shorter than the previous protocol. Quality parameters (conversion rate, call rate, reproducibility and concordance) were at the same levels as when genotype calls were acquired using the previous protocol. Multiplex PCR with 192 pairs of locus-specific primers was available for target preparation in the DigiTag2 assay without the optimization of reaction conditions, and quality parameters had the same levels as those acquired with 96-plex PCR. The locus-specific primers were able to achieve sufficient (concentration of target amplicon ≥5 nM) and specific (concentration of unexpected amplicons <2 nM) amplification within 2 hours, were also able to achieve detectable amplifications even when working in a 96-plex or 192-plex form. The improved DigiTag2 assay will be an efficient platform for screening an intermediate number of SNPs (tens to hundreds of sites) in the replication analysis after genome-wide association study. Moreover, highly parallel and short-acting amplification with locus-specific primers may thus facilitate widespread application to other PCR-based assays

One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method

[EN] An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nm-laser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probedensity and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.3 x 10(8) -8.9 x 10(8) fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis. (C) 2014 Elsevier B.V. All rights reserved.This research has been funded through Projects GVA-PROMETEO/2010/008 (Generalitat Valenciana) and CTQ/2013/ 45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for her PhD studies.Santiago Felipe, S.; Tortajada-Genaro, LA.; Morais, S.; Puchades, R.; Maquieira Catala, Á. (2014). One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method. Sensors and Actuators B: Chemical. 204:273-281. https://doi.org/10.1016/j.snb.2014.07.073S27328120

Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone

Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian intitiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites

Genome‐wide association study of café‐au‐lait macule number in neurofibromatosis type 1

Abstract Background Neurofibromatosis type 1 (NF1) is a tumor‐predisposition disorder that arises due to pathogenic variants in tumor suppressor NF1. NF1 has variable expressivity that may be due, at least in part, from heritable elements such as modifier genes; however, few genetic modifiers have been identified to date. Methods In this study, we performed a genome‐wide association analysis of the number of café‐au‐lait macules (CALM) that are considered a tumor‐like trait as a clinical phenotype modifying NF1. Results A borderline genome‐wide significant association was identified in the discovery cohort (CALM1, N = 112) between CALM number and rs12190451 (and rs3799603, r2 = 1.0; p = 7.4 × 10−8) in the intronic region of RPS6KA2. Although, this association was not replicated in the second cohort (CALM2, N = 59) and a meta‐analysis did not show significantly associated variants in this region, a significant corroboration score (0.72) was obtained for the RPS6KA2 signal in the discovery cohort (CALM1) using Complementary Pairs Stability Selection for Genome‐Wide Association Studies (ComPaSS‐GWAS) analysis, suggesting that the lack of replication may be due to heterogeneity of the cohorts rather than type I error. Conclusion rs12190451 is located in a melanocyte‐specific enhancer and may influence RPS6KA2 expression in melanocytes—warranting further functional studies