Biology subjects

Skevin, S.

Publications and source records attributed to Skevin, S..

3 recordsLinked to original sources

bioRxiv2026

Recombinase polymerase amplification: characterization and mitigation of undescribed multimeric artefacts

Recombinase polymerase amplification (RPA) enables rapid nucleic acid testing in low-resource environments, but poorly characterized byproducts can compromise assay specificity and cause false-positive results. Here, we amplified the thirteen original CODIS core loci and Amelogenin to characterize recurrent RPA artefacts and establish conditions that reduce their formation. First, RPA products were analyzed for two reference samples by Oxford Nanopore Technologies sequencing. This revealed two distinct classes of multimeric products: primer multimers and amplicon multimers, consisting of repeated primer or amplicon sequences, respectively. Individual artefacts contained up to 281 primer copies or 22 amplicon copies, demonstrating the extensive range of these products. Next, we performed an optimization study to evaluate the effects of reaction temperature and reagent concentrations at two representative loci, D3S1358 and D5S818. Among the conditions tested, temperature had the most pronounced effect. Reducing the temperature from 42{degrees}C to 34{degrees}C increased the relative target amplicon fraction from 15% to 83% for D3S1358 and from 84% to 98% for D5S818, while maintaining or increasing absolute target concentration. Lower primer concentrations and higher T4 UvsX concentrations also reduced multimer formation, although lower primer concentrations reduced target yield and caused allelic dropout. Finally, amplification at 34{degrees}C was evaluated across all fourteen loci by sequencing. Relative to 42{degrees}C, the target read fraction increased by more than 5 percentage points for 7/14 loci in one reference sample and 9/14 loci in the other, with the largest improvements at multimer-prone loci. These findings identify multimers as an important class of RPA artefacts and establish reaction temperature and T4 UvsX concentration as promising conditions to improve RPA specificity.

biochemistry↗

bioRxiv2025

Recombinase Polymerase Amplification of Forensic Short Tandem Repeat Loci

Short tandem repeats (STRs) are highly polymorphic repetitive DNA sequences extensively used in forensic science for identification of individuals. STR genotyping is usually performed by capillary electrophoresis (CE) or next-generation sequencing (NGS) in centralized laboratories. However, there is an increasing need for a low cost, portable and rapid STR genotyping method. Multiple methods for miniaturization have been explored, all relying on polymerase chain reaction (PCR) for generating amplicons. PCR requires precise thermal cycling, which complicates the design of the STR genotyping microfluidic device. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification method that operates between 37{degrees}C and 42{degrees}C and completes within 40 minutes. This, along with the robustness of reagents and reduced stutter rate compared to PCR makes RPA a suitable candidate for implementation in an STR genotyping microfluidics device as well as a part of the established STR genotyping work flows. In this proof-of-concept study, we evaluate RPA assay for amplification of forensically relevant STR loci. Thirteen core STR loci of the Combined DNA Index System (CODIS) were amplified using RPA in both singleplex and multiplex formats. The amplicons were then analyzed using three different methods: CE, Illumina and Oxford Nanopore Technologies (ONT). A subset of 5 loci was used for CE analysis. CE, Illumina and ONT sequencing of singleplex RPA each resulted in complete and correct STR profiles across all samples. Sensitivity assessment demonstrated that complete and correct genotypes were achieved with DNA inputs of 62 pg and above for all but locus D8S1179. Attempts at multiplex RPA amplification resulted in incomplete or incorrect STR profiles. This outcome highlights a challenge in adapting RPA for simultaneous amplification of multiple STR loci, which is a standard requirement in forensic DNA profiling.

genomics↗

bioRxiv2022

Transcriptional and translational dynamics underlying heat shock response in the thermophilic Crenarchaeon Sulfolobus acidocaldarius

High-temperature stress is critical for all organisms and induces a profound cellular response. For Crenarchaeota, little information is available on how heat shock affects cellular processes and on how this response is regulated. In this work, we set out to study heat shock response in the thermoacidophilic model crenarchaeon Sulfolobus acidocaldarius, which thrives in volcanic hot springs and has an optimal growth temperature of 75{degrees}C. Pulse-labeling experiments demonstrated that a temperature shift to 86{degrees}C induces a drastic reduction of the transcriptional and translational activity, but that RNA and protein neosynthesis still occurs. By combining RNA sequencing and TMT-labeled mass spectrometry, an integrated mapping of the transcriptome and proteome was performed. This revealed that heat shock causes an immediate change in the gene expression profile, with RNA levels of half of the genes being affected, followed by the more subtle reprogramming of the protein landscape. A limited correlation was observed in differential expression on the RNA and protein level, suggesting that there is a prevalence of post-transcriptional and post-translational regulation upon heat shock. Furthermore, based on the finding that promoter regions of heat shock regulon genes lack a conserved DNA-binding motif, we propose that heat-shock responsive transcription regulation is likely not to be accomplished by a classical transcription factor. Instead, in contrast to histone-harboring Euryarchaeota that have heat-shock transcription factors, it is hypothesized that Sulfolobales and other histone-lacking thermophilic archaea employ an evolutionary ancient mechanism relying on temperature-responsive changes in DNA organization and compaction, induced by the action of nucleoid-associated proteins.

microbiology↗