Enzyme-free optical DNA mapping of the human genome using competitive binding

Abstract

Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized.

Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging

“…Furthermore, the assay allows direct investigation of heterogeneities in the amount of DNA damage along the genome. By combining the damage assay with optical DNA mapping it is possible to identify where along the genome each damage site is located [ 64 ]. This could be combined with commercial systems for optical DNA mapping to investigate genome-wide levels of DNA damage, similar to what was recently demonstrated for the epigenetic markers 5-methylcytosine [ 65 ] and 5-hydroxymethylcytosine [ 66 ].…”

Advances in optical mapping for genomic research

“…In 2019, Müller et al . [133] published a protocol for enzyme-free optical mapping by competitive binding of YOYO-1 and netropsin. Their method blocks an AT-rich region that highlights a DNA contour, preventing DNA damage by nicking.…”

Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale

“…The labeling schemes in optical genome mapping are roughly divided into two main subgroups: sparse labeling, where enzymatic reactions are used to fluorescently tag specific sequences scattered along the long DNA molecules, creating a barcode-like pattern where each marker can be localized [4,23,24]; and continuous labeling, where frequent labeling [25], denaturation mapping [26][27][28] or affinity-based binding [1,27,28] generates a continuous fluorescence intensity map along the molecule [1,27,29]. The maps generated by both methods provide a unique fluorescent signature that discloses the molecule's genomic origin [1,[3][4][5]30,31]. This molecule blocks the dye from binding to these sites, thus keeping them dark while the rest of the genome is labeled, resulting in a sequence-specific intensity profile of the molecules.…”

“…Nyberg et al [ 42 ] used it to identify plasmids in a clinical isolate, and Müller et al combined it with CRISPR/Cas9 to identify antibiotic resistance genes [ 43 ]. Recently, competitive binding was used by Müller et al to map fragments of the human genome [ 30 ]. Nevertheless, the method has not yet been applied for high-throughput mapping of large genomes.…”

“…Next, DNA polymerase and ligase are introduced and fill this gap with fluorescent nucleotides, allowing the visualization of the repaired damage sites ( Figure 4 B,iii). As a proof of principle, sites of DNA damage induced by the chemotherapeutic agent etoposide were recently labeled and mapped using competitive binding [ 30 ]. In the future, optical mapping can potentially be used to reveal the effect of drugs and environmental toxicants on the genome, specifically at regions that are hard to analyze by NGS, and locate genomic hotspots prone to damage and mutations.…”