DNA Methylation: A Timeline of Methods and Applications

DNA methylation is a biochemical process where a DNA base, usually cytosine, is enzymatically methylated at the 5-carbon position. An epigenetic modification associated with gene regulation, DNA methylation is of paramount importance to biological health and disease. Recently, the quest to unravel the Human Epigenome commenced, calling for a modernization of previous DNA methylation profiling techniques. Here, we describe the major developments in the methodologies used over the past three decades to examine the elusive epigenome (or methylome). The earliest techniques were based on the separation of methylated and unmethylated cytosines via chromatography. The following years would see molecular techniques being employed to indirectly examine DNA methylation levels at both a genome-wide and locus-specific context, notably immunoprecipitation via anti-5′methylcytosine and selective digestion with methylation-sensitive restriction endonucleases. With the advent of sodium bisulfite treatment of DNA, a deamination reaction that converts cytosine to uracil only when unmethylated, the epigenetic modification can now be identified in the same manner as a DNA base-pair change. More recently, these three techniques have been applied to more technically advanced systems such as DNA microarrays and next-generation sequencing platforms, bringing us closer to unveiling a complete human epigenetic profile

Replication and exploratory analysis of 24 candidate risk polymorphisms for neural tube defects.

BackgroundNeural tube defects (NTDs), which are among the most common congenital malformations, are influenced by environmental and genetic factors. Low maternal folate is the strongest known contributing factor, making variants in genes in the folate metabolic pathway attractive candidates for NTD risk. Multiple studies have identified nominally significant allelic associations with NTDs. We tested whether associations detected in a large Irish cohort could be replicated in an independent population.MethodsReplication tests of 24 nominally significant NTD associations were performed in racially/ethnically matched populations. Family-based tests of fifteen nominally significant single nucleotide polymorphisms (SNPs) were repeated in a cohort of NTD trios (530 cases and their parents) from the United Kingdom, and case-control tests of nine nominally significant SNPs were repeated in a cohort (190 cases, 941 controls) from New York State (NYS). Secondary hypotheses involved evaluating the latter set of nine SNPs for NTD association using alternate case-control models and NTD groupings in white, African American and Hispanic cohorts from NYS.ResultsOf the 24 SNPs tested for replication, ADA rs452159 and MTR rs10925260 were significantly associated with isolated NTDs. Of the secondary tests performed, ARID1A rs11247593 was associated with NTDs in whites, and ALDH1A2 rs7169289 was associated with isolated NTDs in African Americans.ConclusionsWe report a number of associations between SNP genotypes and neural tube defects. These associations were nominally significant before correction for multiple hypothesis testing. These corrections are highly conservative for association studies of untested hypotheses, and may be too conservative for replication studies. We therefore believe the true effect of these four nominally significant SNPs on NTD risk will be more definitively determined by further study in other populations, and eventual meta-analysis

The application of CRISPR-Cas for single species identification from environmental DNA

We report the first application of CRISPR‐Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows non‐invasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR‐based techniques to detect a given species from eDNA samples, posing a logistical challenge for on‐site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR‐Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof‐of‐concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 h). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity

Evaluation of a point-of-use device used for autoantibody analysis and its potential for following microcystin leucine-arginine exposure

This research article was published in Frontiers in Sensors, Volume 5, 2024Introduction: Globally, the need for measuring exposure to algal toxins has become urgent due to ever-increasing reports of contamination in sea and freshwater, in shellfish and fish stocks and in aerosols. Methods: To address this issue, we evaluated the potential of determining autoantibodies to a panel of biomarkers known to be elevated following exposure to the hepatotoxin microcystin leucine-arginine. The presence of autoantibodies, specific to four selected stress-response, metabolomic and chaperone biomarkers, namely, Heat shock protein 1, Triosephosphate isomerase, Peroxiredoxin 1 and Peroxiredoxin 2 was employed in screening 371 serum samples from microcystin-exposed individuals in Tanzania. In addition, the capacity of the LightDeck fluorescence-based detector, a point-of-use device, to monitor these autoantibody responses in comparison to enzyme-linked immunosorbent assay was evaluated. Results: By using the determination of autoantibodies to this novel panel of biomarkers an altered response was observed following microcystin exposure, with levels generally upregulated. The presence of elevated levels of microcystin leucine-arginine in water, as well as in food sources in Tanzania, may potentially have significant health effects on the population. Discussion: This novel biomarker panel may have potential for the detection of microcystin leucine-arginine exposure as well as various microcystin exposure-associated cancers (e.g., hepatocellular cancer and colorectal cancer). In addition, the utilisation of the LightDeck point-of-use device proved successful for the rapid analysis of this biomarker panel

The differential translation capabilities of the human DHFR2 gene indicates a developmental and tissue specific endogenous protein of low abundance.

A functional role has been ascribed to the human Dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homologue, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2 specific peptides as evidence of its translation. We show definitive evidence that the dihydrofolate reductase activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion, showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, show differential association with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2 specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level

Advice to consider when developing a CRISPR-Cas assay for single species detection using eDNA

Development of simple and rapid techniques to monitor species of conservation importance is vital to further the capabilities of environmental DNA. Conventional methods for eDNA detection pose a logistical challenge for on-site monitoring due to the need for high temperatures and thermal cycling. To circumvent this, we recently adapted an isothermal CRISPR-Cas based detection assay for single-species assessment of Salmo salar as a route to a simple, cost-effective biosensor device (Williams et al., 2019).CRISPR-Cas for detection (rather than genome editing) was first developed for clinical diagnostic applications. The variety of Cas nucleases allow detection of either RNA or DNA with attomolar sensitivity (Chen et al., 2018; Gootenberg et al., 2017). This detection approach is versatile and has recently been adopted for the detection of SARS-CoV-2 (Broughton et al., 2020). The CRISPR-Cas detection system consists of two main elements; a guide RNA specific to the target and an effector Cas12a nuclease. The Cas12a nuclease will only cleave at the target site when a specific protospacer adjacent motif (PAM) is present downstream. The requirement to recognise two separate sequences supports a highly specific recognition system that can distinguish closely related species. However, although its use is expanding rapidly for the detection of pathogens, it is yet to be fully embraced for eDNA detection. The RPA-CRISPR-Cas methodology we have developed utilises the isothermal recombinase polymerase amplification and CRISPR-Cas12a detection, leading to four unique sequence recognition elements, which require stringent design and in-lab testing to ensure assay specificity. Development of our published S. salar CRISPR-Cas assay (Williams et al., 2019), and subsequent assays for Salmo trutta and Salvelinus alpinus, highlighted critical steps to consider and pitfalls to avoid when designing such isothermal assays.1) Only the target sequence should contain the required PAM site.In version 1 of our assay, both S. salar and S. trutta contained the PAM site; we were unable to distinguish them.2) An RPA primer screen is essential.Multiple forward and reverse primers are screened up-/down-stream of the gRNA binding region to select the optimum primer pair.3) Specificity tests should be carried out on tissue from the target species and other species present in the sampling environment.In silico design is not sufficient to ensure assay specificity. ReferencesBroughton, J. P., Deng, X., Yu, G., Fasching, C. L., Servellita, V., Singh, J., … Chiu, C. Y. (2020). CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology, 38(7). https://doi.org/10.1038/s41587-020-0513-4Chen, J. S., Ma, E., Harrington, L. B., Costa, M. Da, Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(6387), 436–439. https://doi.org/10.1126/SCIENCE.AAR6245Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., … Zhang, F. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 356(6336), 438–442. https://doi.org/10.1126/science.aam9321Williams, M. A., O’Grady, J., Ball, B., Carlsson, J., de Eyto, E., McGinnity, P., … Parle-McDermott, A. (2019). The application of CRISPR-Cas for single species identification from environmental DNA. Molecular Ecology Resources, 19(5). https://doi.org/10.1111/1755-0998.1304

Comparing CRISPR-Cas and qPCR eDNA assays for the detection of Atlantic salmon (Salmo salar L.)

Molecular techniques offer sensitive, specific, noninvasive monitoring of target species from a variety of environmental samples. We recently developed a CRISPR‐Cas‐based eDNA assay for rapid single‐species detection as a route to a simple, cost‐effective biosensor device. CRISPR‐Cas‐based diagnostic assays use isothermal conditions in combination with a highly specific sequence recognition system. This CRISPR‐Cas assay was designed to target Salmo salar, and we previously demonstrated its utility in eDNA samples from sites in Ireland. The aim of this study was to validate our assay in two larger sample sets from Canada (n = 16/n = 63) in comparison with an independent S. salar qPCR assay. We demonstrate that overall, the CRISPR‐Cas assay performs similarly to qPCR for assessing the presence or absence of S. salar from eDNA and provides a viable alternative approach where qPCR assay design and application have proven to be challenging