A dual-reference study design for understanding and improving AAV genome size analysis.

Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89 pg/µL (3 × 1010 GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at ∼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.

Methylene Blue-Stained Single-Stranded DNA Aptamers as a Highly Efficient Electronic Switch for Quasi-Reagentless Exosomes Detection: An Old Dog with New Tricks.

Improving the convenience, sensitivity, and cost-effectiveness of electrochemical biosensors is crucial for advancing their clinical diagnostic applications. Herein, we presented an elegant approach to construct electrochemical aptasensors for tumor-derived exosome detection by harnessing the alterable interaction between methylene blue (MB) and DNA aptamer. In detail, the anti-EpCAM aptamer, named SYL3C, was found to exhibit a strong affinity toward MB due to the specific interaction between MB and unbound guanine bases. Thereby, SYL3C could be stained with MB to arouse a strong electrochemical signal on a gold electrode (AuE). Upon binding to EpCAM-positive exosomes, SYL3C underwent a conformational transformation. The resulting conformation, or exosomes-SYL3C complex, not only reduced the accumulation of MB on SYL3C by obstructing the accessibility of guanines to MB but also impeded the transfer of electrons from the bound MB to AuE, leading to a notable decrease in the electrochemical signal. Using MB-stained SYL3C as an electronic switch, an electrochemical aptasensor was readily established for the detection of EpCAM-positive exosome detection. Without the need for signal amplification strategies, expensive auxiliary reagents, and complex operation, this unique signal transduction mechanism alone could endow the aptasensor with ultrahigh sensitivity. A limit of detection (LOD) of 234 particles mL-1 was achieved, surpassing the performance of most reported methods. As a proof of concept, the aptasensor was applied to analyze clinical serum samples and effectively distinguish non-small-cell lung cancer (NSCLC) patients from healthy individuals. As EpCAM exhibits broad expression in exosomes derived from different tumor sources, the developed aptasensor holds promise for diagnosing other tumor types.

Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ.

Transcription is a highly dynamic process that generates single-stranded DNA (ssDNA) in the genome as 'transcription bubbles'. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, based on the fast and specific reaction between N3-kethoxal and guanines in ssDNA. KAS-seq allows rapid (within 5 min), sensitive and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ using as few as 1,000 cells. KAS-seq enables definition of a group of enhancers that are single-stranded and enrich unique sequence motifs. These enhancers are associated with specific transcription-factor binding and exhibit more enhancer-promoter interactions than typical enhancers do. Under conditions that inhibit protein condensation, KAS-seq uncovers a rapid release of RNA polymerase II (Pol II) from a group of promoters. KAS-seq thus facilitates fast and accurate analysis of transcription dynamics and enhancer activities simultaneously in both low-input and high-throughput manner.

New DNA-hydrolyzing DNAs isolated from an ssDNA library carrying a terminal hybridization stem.

DNA-hydrolyzing DNAs represent an attractive type of DNA-processing catalysts distinctive from the protein-based restriction enzymes. The innate DNA property has enabled them to readily join DNA-based manipulations to promote the development of DNA biotechnology. A major in vitro selection strategy to identify these DNA catalysts relies tightly on the isolation of linear DNAs processed from a circular single-stranded (ss) DNA sequence library by self-hydrolysis. Herein, we report that by programming a terminal hybridization stem in the library, other than the previously reported classes (I & II) of deoxyribozymes, two new classes (III & IV) were identified with the old selection strategy to site-specifically hydrolyze DNA in the presence of Zn2+. Their representatives own a catalytic core consisting of ∼20 conserved nucleotides and a half-life of ∼15 min at neutral pH. In a bimolecular construct, class III exhibits unique broad generality on the enzyme strand, which can be potentially harnessed to engineer DNA-responsive DNA hydrolyzers for detection of any target ssDNA sequence. Besides the new findings, this work should also provide an improved approach to select for DNA-hydrolyzing deoxyribozymes that use various molecules and ions as cofactors.

Improving the detection limit of Salmonella colorimetry using long ssDNA of asymmetric-PCR and non-functionalized AuNPs.

A colorimetric sensor based on gold nanoparticles (AuNPs) and single-stranded DNA (ssDNA) is a simple and rapid method for detecting foodborne pathogens. However, the colorimetric method employed in previous studies involved short ssDNA (<100 nucleotides), including the aptamer and PCR products, resulting in the high detection limit of this technique. In this study, a colorimetric sensor was developed based on long ssDNA of asymmetric PCR (aPCR) and non-functionalized AuNPs for detecting Salmonella Typhimurium (S. Typhimurium). In the presence of S. Typhimurium, the long ssDNA (547 nt) amplified by aPCR-protected AuNPs from NaCl-induced aggregation, while the solution retained a red color. After optimizing parameters, the limit of detection (LOD) of the colorimetric sensor was 2.56 CFU/mL with high specificity. Recovery studies showed its feasibility for detecting S. Typhimurium (102 CFU/mL, 104 CFU/mL, and 106 CFU/mL) in spiked lettuce samples. This colorimetric sensor provides new opportunities for the highly sensitive detection of bacteria in real food samples.

Testing a series of modifications on genomic library preparation methods for ancient or degraded DNA.

Technological advancements have revolutionized ancient and degraded DNA analysis, moving the field to the Next Generation Sequencing era. One of the advancements, the ancient DNA-oriented high-throughput library preparation methods, enabled the sequencing of more endogenous molecules. Although fairly optimized, both single- and double-stranded library preparation methods hold the potential for further improvement. Here, we test a series of modifications made at different steps of both single- and double-stranded library preparation methods. Given all the modifications tested, we found that two of them provide further benefits, including the use of Endonuclease VIII as a pre-treatment step before preparing single-stranded libraries and the use of a modified second adapter of the single stranded-libraries as an alternative option to enable sequencing of single stranded-libraries with the standard Illumina sequencing primer instead of the custom designed as described in the single stranded library preparation method. Furthermore, we propose uracil-DNA-glycosylase (UDG) could also be considered for both single- and double-stranded library preparation methods, although additional parameters should be taken into account depending on the sequencing strategy and the sample characteristics. Further modifications were also tested and although they were not advantageous, they could be considered as equivalent to the published options.

PPV and Detection Rate of mt-sDNA Testing, FIT, and CT Colonography for Advanced Neoplasia: A Hierarchic Bayesian Meta-Analysis of the Noninvasive Colorectal Screening Tests.

BACKGROUND. Noninvasive tests for colorectal cancer (CRC) screening and prevention limit the need for invasive colonoscopy to follow up positive test results. However, the relative performance characteristics of available noninvasive tests have not yet been adequately compared. OBJECTIVE. We performed a systematic review and meta-analysis to compare the diagnostic performance of the available noninvasive CRC screening tests, including multitarget stool DNA (mt-sDNA) testing, fecal immunochemical testing (FIT), and CT colonography (CTC), with an emphasis on comparison of PPV and detection rate (DR) for advanced neoplasia (AN; encompassing cases of advanced adenomas and CRC). EVIDENCE ACQUISITION. After systematic searches of MEDLINE and Google Scholar databases, 10 mt-sDNA, 27 CTC, and 88 FIT published screening studies involving 25,132, 33,493, and 2,355,958 asymptomatic adults, respectively, were included. Meta-analysis with hierarchic Bayesian modeling was conducted in accordance with Cochrane Collaboration and PRISMA guidelines to determine test positivity rates (TPRs) leading to optical colonoscopy, as well as PPVs and DRs for both AN and CRC. Different positivity thresholds were considered for FIT and CTC. EVIDENCE SYNTHESIS. Point estimates (with 95% credible intervals) from pooled Bayesian meta-analysis combining all thresholds for FIT and stratifying CTC results by a polyp size threshold of 6 mm or larger (CTC6) and 10 mm or larger (CTC10) were calculated. TPR was 13.5% (10.9-16.6%) for mt-sDNA testing, 6.4% (5.8-7.2%) for FIT, 13.4% (11.4-15.6%) for CTC6, and 6.6% (5.2-7.7%) for CTC10. AN PPV was 26.9% (95% credible interval, 21.8-33.2%) for mt-sDNA testing, 31.8% (29.3-34.5%) for FIT, 34.4% (27.2-41.0%) for CTC6, and 61.0% (54.0-70.0%) for CTC10. CRC PPV was 2.4% (1.5-3.9%) for mt-sDNA testing, 4.9% (4.3-5.3%) for FIT, 3.5% (2.5-4.8%) for CTC6, and 6.0% (4.3-8.0%) for CTC10. The DR for AN was 3.4% (95% credible interval, 2.5-4.8%) for mt-SDNA, 2.0% (1.8-2.3%) for FIT, 4.8% (4.0-6.5%) for CTC6, and 4.0% (3.0-4.6%) for CTC10. When FIT is restricted to a lower threshold (< 10 µg Hb/g feces), its performance profile is similar to that of mt-sDNA testing, although available data are limited. AN PPV odds ratios (relative to CTC10 as the reference) were 0.24 (95% credible interval, 0.17-0.33) for mt-sDNA testing, 0.30 (0.24-0.45) for FIT, and 0.33 (0.25-0.47) for CTC6. CONCLUSION. Among noninvasive CRC screening tests, CTC with a polyp size threshold of 10 mm or larger most effectively targets AN, preserving detection while also decreasing unnecessary colonoscopies compared with mt-sDNA testing and FIT. CLINICAL IMPACT. CTC performed with a polyp size threshold for colonoscopy referral set at 10 mm or larger represents the most effective and efficient noninvasive screening test for CRC prevention and detection.

Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA.

Detection of cell-free DNA (cfDNA) has an impact on DNA analysis in liquid biopsies. However, current strategies to detect cfDNA have limitations that should be overcome, such as having low sensitivity and requiring much time and a specialized instrument. Thus, non-invasive and rapid detection tools are needed for disease prevention and early-stage treatment. Here we developed a device having a microheater integrated with zinc oxide nanowires (microheater-ZnO-NWs) to detect target single-stranded DNAs (ssDNAs) based on DNA probe hybridization. We confirmed experimentally that our device realizedin-situannealed DNA probes by which we subsequently detected target ssDNAs. We envision that this device can be utilized for fundamental studies related to nanobiodevice-based DNA detection.

Gold nanoparticle-assisted plasmonic enhancement for DNA detection on a graphene-based portable surface plasmon resonance sensor.

The impact of different gold nanoparticle (GNP) structures on plasmonic enhancement for DNA detection is investigated on a few-layer graphene (FLG) surface plasmon resonance (SPR) sensor. Two distinct structures of gold nano-urchins (GNu) and gold nanorods (GNr) were used to bind the uniquely designed single-stranded probe DNA (ssDNA) of Mycobacterium tuberculosis complex DNA. The two types of GNP-ssDNA mixture were adsorbed onto the FLG-coated SPR sensor through the π-π stacking force between the ssDNA and the graphene layer. In the presence of complementary single-stranded DNA, the hybridization process took place and gradually removed the probes from the graphene surface. From SPR sensor preparation, the annealing process of the Au layer of the SPR sensor effectively enhanced the FLG coverage leading to a higher load of the probe DNA onto the sensing interface. The FLG was shown to be effective in providing a larger surface area for biomolecular capture due to its roughness. Carried out in the DNA hybridization study with the SPR sensor, GNu, with its rough and spiky structures, significantly reinforced the overall DNA hybridization signal compared with GNr with smooth superficies, especially in capturing the probe DNA. The DNA hybridization detection assisted by GNu reached the femtomolar range limit of detection. An optical simulation validated the extreme plasmonic field enhancement at the tip of the GNu spicules. The overall integrated approach of the graphene-based SPR sensor and GNu-assisted DNA detection provided the proof-of-concept for the possibility of tuberculosis disease screening using a low-cost and portable system to be potentially applied in remote or third-world countries.

Electrochemical detection of alkaline phosphatase activity through enzyme-catalyzed reaction using aminoferrocene as an electroactive probe.

As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100 mU/mL with the limit of detection (LOD) of 1.48 mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".

DNA immobilization and detection using DNA binding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNA binding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNA binding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNA binding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNA binding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNA binding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.

DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute.

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

Spectrochemical Probing of MicroRNA Duplex Using Spontaneous Raman Spectroscopy for Biosensing Applications.

MicroRNAs are emerging as both diagnostic and therapeutic targets in different human pathologies. An accurate understanding of the structural dependency of microRNAs for their biological functions is essential for designing synthetic oligos with various base and linkage modifications that can transform into highly sensitive diagnostic devices and therapeutic molecules. In this proof-of-principle study, we have utilized label-free spontaneous Raman spectroscopy to understand the structural differences in sense and antisense microRNA-21 by hybridizing them with complementary RNA and DNA oligos. Overall, the results suggest that the changes in the Raman band at 785 cm-1 originating from the phosphodiester bond of the nucleic acid backbone, linking 5' phosphate of the nucleic acid with 3' OH of the other nucleotide, can serve as a marker to identify these structural variations. Our results support the application of Raman spectroscopy in discerning intramolecular (ssRNA and ssDNA) and intermolecular (RNA-RNA, RNA-DNA, and DNA-DNA hybrids) interactions of nucleic acids. This is potentially useful for developing biosensors to quantify microRNAs in clinical samples and to design therapeutic microRNAs with robust functionality.

Experimental Comparison in Sensing Breast Cancer Mutations by Signal ON and Signal OFF Paper-Based Electroanalytical Strips.

The development of paper-based electroanalytical strips as powerful diagnostic tools has gained a lot of attention within the sensor community. In particular, the detection of nucleic acids in complex matrices represents a trending topic, especially when focused toward the development of emerging technologies, such as liquid biopsy. DNA-based biosensors have been largely applied in this direction, and currently, there are two main approaches based on target/probe hybridization reported in the literature, namely Signal ON and Signal OFF. In this technical note, the two approaches are evaluated in combination with paper-based electrodes, using a single strand DNA relative to H1047R (A3140G) missense mutation in exon 20 in breast cancer as the model target. A detailed comparison among the analytical performances, detection protocol, and cost associated with the two systems is provided, highlighting the advantages and drawbacks depending on the application. The present work is aimed to a wide audience, particularly for those in the field of point-of-care, and it is intended to provide the know-how to manage with the design and development stages, and to optimize the platform for the sensing of nucleic acids using a paper-based detection method.

Open-tubular nanoelectrochromatography (OT-NEC): gel-free separation of single stranded DNAs (ssDNAs) in thermoplastic nanochannels.

Electrophoresis or electrochromatography carried out in nanometer columns (width and depth) offers some attractive benefits compared to microscale columns. These advantages include unique separation mechanisms that are scale dependent, fast separation times, and simpler workflow due to the lack of a need for column packing and/or wall coatings to create a stationary phase. We report the use of thermoplastics, in this case PMMA, as the substrate for separating single-stranded DNAs (ssDNAs). Electrophoresis nanochannels were created in PMMA using nanoimprint lithography (NIL), which can produce devices at lower cost and in a higher production mode compared to the fabrication techniques required for glass devices. The nanochannel column in PMMA was successful in separating ssDNAs in free solution that was not possible using microchip electrophoresis in PMMA. The separation could be performed in <1 s with resolution >1.5 when carried out using at an electric field strength of 280 V/cm and an effective column length of 60 µm (100 nm × 100 nm, depth and width). The ssDNAs transport through the PMMA column was driven electrokinetically under the influence of an EOF. The results indicated that the separation was dominated by chromatographic effects using an open tubular nano-electrochromatography (OT-NEC) mode of separation. Interesting to these separations was that no column packing was required nor a wall coating to create the stationary phase; the separation was affected using the native polymer that was UV/O3 activated and an aqueous buffer mobile phase.

Sperm chromatin condensation and single- and double-stranded DNA damage as important parameters to define male factor related recurrent miscarriage.

The aim of the present work is to characterize the relationship between sperm protamine deficiency and single- and double-stranded DNA damage and to assess the diagnostic potential of chromomycin A3 (CMA3). For that purpose, semen samples from 90 human males with different clinical features were included (fertile donors, patients with recurrent pregnancy loss [RPL], and infertile patients). DNA condensation was analyzed by CMA3 and different types of DNA fragmentation were analyzed through the comet assay. A positive correlation between DNA condensation and single-stranded DNA fragmentation was found (Rs = .456; p = .05). CMA3 presented differences between fertile donors and all other groups (p < .001). Interestingly, patients with RPL, who were able to achieve a pregnancy, and infertile patients showed similar values of CMA3 (p > .05). Receiver operating characteristic curves and the profiles obtained by the combination of Comet assays and CMA3 indicate that the CMA3 test may be an interesting approach to distinguish those subjects with higher pregnancy loss risk from fertile donors (CMA3 area under the curve 0.928, with a confidence interval of 0.849-1.000). The present work shows that DNA condensation is related to oxidative damage, which affects mainly protamine-rich regions. The profiles observed in different clinical groups showed that CMA3 might be useful for the diagnosis of RPL risk when combined with Comet assays.

DNA detection by SERS: hybridisation parameters and the potential for asymmetric PCR.

The use of surface enhanced Raman scattering (SERS) for the detection of DNA has significant potential in terms of sensitivity, multiplex target detection and robust signal detection from nanoparticles. Current methods are usually performed with short chain DNA fragments or require additional separation steps for detection of longer chain fragments. We present an integrated method for the rapid and sensitive detection of extended (≥100-base) nucleic acids with reduced preparation and sample separation steps. Key to this is the DNA sequence-specific assembly of silver nanoparticles labelled with a Raman tag to provide an enhanced signal from the tag and hence molecular recognition of the target DNA. DNA probe orientation and hybridisation procedures are critical for the success of this assay. Effective detection of extended nucleic acids was achieved with head-to-head probes and by adding polyethylene glycol 10 000 (PEG 10 000) to the hybridisation buffer. This gave a 34-fold discriminatory enhancement factor when applied to a synthetic target. A structured approach toward maximising hybridisation procedures and SERS response is described, followed by an initial demonstration of SERS detection of single-stranded DNA target amplified by asymmetric PCR which was used without further separation. This has implications for future developments in using SERS for DNA detection due to the new-found ability to integrate SERS with asymmetric PCR.

Wafer-level fabrication of individual solid-state nanopores for sensing single DNAs.

For biomolecule sensing purposes a solid-state nanopore platform based on silicon has certain advantages as compared to nanopores on other substrates such as graphene, silicon nitride, silicon oxide etc Capitalizing on the developed CMOS technology, nanopores on silicon are scalable without any requirement for additional processing, the devices are low cost and the process can be repeatable with a high yield. One of the essential requirements in biomolecule sensing is the ability of the nanopore to interact with the analyte. In this work, we present a method for processing high aspect ratio, single nanopores in the range of 10-30 nm in diameter and approximately 700 nm in length on a silicon-on-insulator (SOI) wafer. The presented method of manufacturing the high aspect ratio individual nanopores combines optical lithography and anisotropic KOH etching with a final electrochemical etching step to form the nanopores and is repeatable and can be processed in batches. We demonstrate electrical detection of dsDNA translocation, where the characteristic time of the process is in the millisecond range. We also analyse the translocation parameters and correlate the enhanced length of the nanopore to a longer translocation time as compared to other substrates.

Evaluating digital PCR for the quantification of human nuclear DNA: determining target strandedness.

Digital polymerase chain reaction (dPCR) methodology has been asserted to be a "potentially primary" analytical approach for assigning DNA concentration. The essence of dPCR measurements is the independent dispersal of fragments into multiple reaction partitions, amplifying fragments containing a target nucleotide sequence until the signal from all partitions containing at least one such fragment rises above threshold, and then determining the proportion of partitions with an above-threshold signal. Should originally double-stranded DNA (dsDNA) fragments be converted into two single strands (ssDNA) prior to dispersal, the dPCR measurements could be biased high by as much as a factor of two. Realizing dPCR's metrological potential therefore requires analytical methods for determining the proportion of ssDNA in nominally dsDNA samples. To meet this need, we have investigated several approaches to this determination: A260 ratio, dPCR ratio, cdPCR staircase, and ddPCR enzyme. In our hands, only the endonuclease-based approach provides adequately accurate estimates for relatively small ssDNA proportions. We present four (enzyme, assay) pairs that provide self-consistent results for human nuclear DNA extracts. However, the proportion of ssDNA differs by as much as 50% between assays, apparently related to the guanine-cytosine (GC) content of the fragment near the assay's target sequence. While materials extracted by us have no more than 6% ssDNA content even after long storage, a commercially obtained PCR assay calibrant contains ≈18% ssDNA. Graphical abstract.

Northern lights assay: a versatile method for comprehensive detection of DNA damage.

DNA damage assays have various limitations in types of lesions detected, sensitivity, specificity and samples that can be analyzed. The Northern Lights Assay (NLA) is based on 2D Strandness-Dependent Electrophoresis (2D-SDE), a technique that separates nucleic acids based on length, strandness, structure and conformation changes induced by damage. NLA is run on a microgel platform in 20-25 min. Each specimen is analyzed in pairs of non-digested DNA to detect single- and double-stranded breaks (DSBs) and Mbo I-digested DNA to detect other lesions. We used NLA to evaluate DNA in solution and isolated from human cells treated with various genotoxic agents. NLA detected and distinguished between single- and DSBs, interstrand and intrastrand DNA crosslinks, and denatured single-stranded DNA. NLA was sufficiently sensitive to detect biologically relevant amount of DNA damage. NLA is a versatile, sensitive and simple method for comprehensive and simultaneous analysis of multiple types of damage, both in purified DNA and in DNA isolated from cells and body fluids. NLA can be used to evaluate DNA quality in biosamples, monitor complex molecular procedures, assess genotoxicity, diagnose genome instability, facilitate cancer theranostics and in basic nucleic acids research.