Single DNA Research Articles

A quantifiler™ trio-based HRM screening assay for the accurate prediction of single source versus mixed biological samples.

At present, the forensic DNA workflow is not capable of providing information about the contributor status (single source vs. multiple contributors) of evidentiary samples prior to end-point analysis. This exacerbates the challenges inherent to mixtures and low-template DNA samples. If additional sample information could be provided earlier in the workflow, protocols could be implemented to mitigate these challenges. An integrated Quantiplex®- high resolution melt (HRM) assay was shown to be effective in distinguishing between single source and mixture DNA samples; however, integration of the HRM assay into a more commonly used chemistry would be beneficial to the practitioner community. Thus, the assay was redesigned as an integrated Quantifiler™ Trio-HRM assay, which included the identification of a new DNA-binding dye, an increased reaction volume, and the establishment of new data analysis and standard curve metrics for all targets. This redesigned assay produced quantification values and qualitative values that were comparable to those produced when the same samples were tested using the standard Quantifiler™ Trio chemistry and settings. Further, STR profiles generated with quantification values produced from the integrated Quantifiler™ Trio-HRM assay and standard Quantifiler™ Trio chemistry were complete and fully concordant. Most importantly, the integrated Quantifiler™ Trio-HRM assay was able to accurately predict whether a sample was single source or a mixture 79.2% of the time, demonstrating the potential of this approach. With the incorporation of an expanded training set for prediction modeling, and completion of critical developmental validation studies, this assay could prove useful to the forensic DNA practitioner community.

ATAC-Me simultaneously decodes chromatin accessibility and DNA methylation.

ATAC-Me probes chromatin accessibility and DNA methylation from a single DNA library and is a powerful method for identifying DNA methylation within accessible regions. Plant nuclei are extracted and incubated with Tn5 transposases that assemble methylated adapters. The accessible regions are tagged by the methylated adapters. The obtained DNA fragments are enriched and treated with sodium bisulfite to convert non-methylated cytosine to thymine. Since the methylated adapters are not converted by sodium bisulfite, these DNA fragments are readily amplified by specific sequencing primers during library construction. After sequencing, accessible regions and DNA methylation can be simultaneously identified by bioinformatics tools.

Dual signal amplification-integrated single-molecule biosensing of flap endonuclease 1 in breast cancer tissues

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that may act as a valuable biomarker for cancer diagnosis. Herein, we construct a dual signal amplification-integrated single-molecule fluorescent biosensor for ultrasensitive detection of FEN1 in breast cancer tissues. We design a triplex DNA containing a flap structure for both recognizing and amplifying FEN1 signal. When FEN1 is present, flap is specifically cleaved by FEN1 and released from triplex DNA to form a short ssDNA strand with free 3' OH terminus, and the resultant ssDNA can serve as the primer and binds to circular template in triplex DNA to trigger rolling circle amplification (RCA), generating a long ssDNA product with abundant repeated activator sequences. The hybridization of the resultant activators with crRNAs subsequently activates Cas12a to catalyze trans-cleavage of Cy5- and BHQ-labeled probes to restore Cy5 fluorescence that can be quantified by single-molecule detection. Notably, only a single triplex DNA is required for both initiating and implementing RCA, greatly simplifying the reaction system and eliminating nonspecific amplification. This biosensor exhibits ultrahigh sensitivity with a detection limit of 2.24 × 10-5 U/μL, facilitating accurate detection of endogenous FEN1 activity with single-cell sensitivity and discrimination of different FEN1 levels in clinical breast cancer tissues.

Oligonucleotide Fluorescence In Situ Hybridization: An Efficient Chromosome Painting Method in Plants.

Fluorescence in situ hybridization (FISH) is an indispensable technique for studying chromosomes in plants. However, traditional FISH methods, such as BAC, rDNA, tandem repeats, and distributed repetitive sequence probe-based FISH, have certain limitations, including difficulties in probe synthesis, low sensitivity, cross-hybridization, and limited resolution. In contrast, oligo-based FISH represents a more efficient method for chromosomal studies in plants. Oligo probes are computationally designed and synthesized for any plant species with a sequenced genome and are suitable for single and repetitive DNA sequences, entire chromosomes, or chromosomal segments. Furthermore, oligo probes used in the FISH experiment provide high specificity, resolution, and multiplexing. Moreover, oligo probes made from one species are applicable for studying other genetically and taxonomically related species whose genome has not been sequenced yet, facilitating molecular cytogenetic studies of non-model plants. However, there are some limitations of oligo probes that should be considered, such as requiring prior knowledge of the probe design process and FISH signal issues with shorter probes of background noises during oligo-FISH experiments. This review comprehensively discusses de novo oligo probe synthesis with more focus on single-copy DNA sequences, preparation, improvement, and factors that affect oligo-FISH efficiency. Furthermore, this review highlights recent applications of oligo-FISH in a wide range of plant chromosomal studies.

Evaluating the cost-effectiveness of low-level HBV DNA screening in occult hepatitis B infection donors: A study from Shandong Blood Center, China

ObjectiveThis study aimed to assess the efficacy of individual-donation nucleic acid testing (ID-NAT) in detecting occult hepatitis B virus infection (OBI) among anti-HBc positive blood donors, compared to minipool nucleic acid testing (MP-NAT). MethodsThe present study analyzed data from the Shandong Blood Center in China during the period from January 2018 to June 2022, where HBsAg-negative blood donors were screened using the 6-sample minipool nucleic acid testing (6-sample MP NAT) method. NAT-positive samples underwent subsequent anti-HBc and anti-HBs testing. Approximately 5000 samples that passed the nucleic acid mixing test were randomly selected for anti-HBc testing, and over 100 anti-HBc positive samples underwent individual donor nucleic acid testing (ID-NAT). Any HBV DNA positive samples detected by ID-NAT were subsequently confirmed using alternative nucleic acid testing methods. ResultsAmong 220,445 HBsAg-negative blood donors, the positivity rate of HBV DNA detection using the 6-sample minipool nucleic acid testing (MP NAT) method was found to be 0.031% (69/220,445). Of the 67 HBV DNA positive samples, 55 (82.09%) and 25 (37.31%) were found to be positive for anti-HBc and anti-HBs, respectively, using the supplementary chemiluminescent microparticle immunoassay (CMIA). Among the 4797 HBsAg-negative/MP NAT-negative samples, 909 (18.95%) tested positive for anti-HBc. Further NAT testing was performed on 164 arbitrarily selected anti-HBc-positive/MP HBV DNA-negative samples, revealing a HBV DNA positivity rate of 1.22% (2/164). ConclusionUsing individual donation nucleic acid testing can significantly increase the detection rate of occult hepatitis B virus infection in anti-HBc-positive blood donors, resulting in a detection rate of 0.22% (1.22 × 0.1895). This rate is 8.10 times higher than the detection rate achieved by mixed testing methods (0.031%) [calculated as (0.22 + 0.031)/0.031]. Therefore, it is recommended to perform single HBV DNA testing on anti-HBc-positive blood donors, discard plasma with weakly positive or negative anti-HBs but positive anti-HBc, or avoid transfusing anti-HBc-positive plasma to recipients with weakly positive or negative anti-HBs to prevent HBV infection.

Detection of long mRNA sequences by a Y-shaped DNA probe with three target-binding segments

Tumor-related mRNA detection is significant and interesting. The current mRNA detection method has the challenge of quantifying long mRNA sequences. Herein, a Y-shaped DNA probe with three target-binding segments was developed to detect tumor-related mRNA. This Y-shaped DNA probe (Y-probe) was assembled by six single DNA strands. Among these DNA strands, two DNA strands contained the split G-quadruplex sequence, and two DNA strands were modified with a pair of fluorophore and quencher, which were used to produce the detectable signal. In the presence of a long target mRNA sequence, target mRNA was hybridized with the three target-binding segments of the Y-probe, resulting in the increased fluorescence of G-quadruplex specific dye Thioflavin T and the decreased fluorescence of fluorophore, which could achieve the ratio detection of target mRNA. The Y-probe exhibited a low detection limit of 17.53 nM. Moreover, this probe showed high accuracy due to the benefits of three target-binding segments.

Molecular system for an exponentially fast growing programmable synthetic polymer

In this paper, we demonstrate a molecular system for the first active self-assembly linear DNA polymer that exhibits programmable molecular exponential growth in real time, also the first to implement “internal” parallel insertion that does not rely on adding successive layers to “external” edges for growth. Approaches like this can produce enhanced exponential growth behavior that is less limited by volume and external surface interference, for an early step toward efficiently building two and three dimensional shapes in logarithmic time. We experimentally demonstrate the division of these polymers via the addition of a single DNA complex that competes with the insertion mechanism and results in the exponential growth of a population of polymers per unit time. In the supplementary material, we note that an “extension” beyond conventional Turing machine theory is needed to theoretically analyze exponential growth itself in programmable physical systems. Sequential physical Turing Machines that run a roughly constant number of Turing steps per unit time cannot achieve an exponential growth of structure per time. In contrast, the “active” self-assembly model in this paper, computationally equivalent to a Push-Down Automaton, is exponentially fast when implemented in molecules, but is taxonomically less powerful than a Turing machine. In this sense, a physical Push-Down Automaton can be more powerful than a sequential physical Turing Machine, even though the Turing Machine can compute any computable function. A need for an “extended” computational/physical theory arises, described in the supplementary material section S1.

CimpleG: finding simple CpG methylation signatures

DNA methylation signatures are usually based on multivariate approaches that require hundreds of sites for predictions. Here, we propose a computational framework named CimpleG for the detection of small CpG methylation signatures used for cell-type classification and deconvolution. We show that CimpleG is both time efficient and performs as well as top performing methods for cell-type classification of blood cells and other somatic cells, while basing its prediction on a single DNA methylation site per cell type. Altogether, CimpleG provides a complete computational framework for the delineation of DNAm signatures and cellular deconvolution.

Programming the morphology of DNA origami crystals by magnesium ion strength

Harnessing the programmable nature of DNA origami for controlling structural features in crystalline materials affords opportunities to bring crystal engineering to a remarkable level. However, the challenge of crystallizing a single type of DNA origami unit into varied structural outcomes remains, given the requirement for specific DNA designs for each targeted structure. Here, we show that crystals with distinct equilibrium phases and shapes can be realized using a single DNA origami morphology with an allosteric factor to modulate the binding coordination. As a result, origami crystals undergo phase transitions from a simple cubic lattice to a simple hexagonal (SH) lattice and eventually to a face-centered cubic (FCC) lattice. After selectively removing internal nanoparticles from DNA origami building blocks, the body-centered tetragonal and chalcopyrite lattice are derived from the SH and FCC lattices, respectively, revealing another phase transition involving crystal system conversions. The rich phase space was realized through the de novo synthesis of crystals under varying solution environments, followed by the individual characterizations of the resulting products. Such phase transitions can lead to associated transitions in the shape of the resulting products. Hexagonal prism crystals, crystals characterized by triangular facets, and twinned crystals are observed to form from SH and FCC systems, which have not previously been experimentally realized by DNA origami crystallization. These findings open a promising pathway toward accessing a rich phase space with a single type of building block and wielding other instructions as tools to develop crystalline materials with tunable properties.

First Report of Powdery Mildew Caused by Podosphaera macularis on Humulus scandens in China.

Humulus scandens, (Lour.) Merr., is a climbing herb which are used as traditional Chinese medicine, a raw material for papermaking, making soap, and replacing hops H. lupulus. This herb is distributed in many provinces of China, including Sichuan province. During March and June 2022, powdery mildew was found on leaves of H. scandens in the modern agricultural high-tech demonstration garden of Sichuan Academy of Agricultural Science, Chengdu, Sichuan Province, China. Abundant white or grayish powdery colonies could be seen on the surface of leaves, and 30%-100% of leaf areas were affected. Some of the infected leaves were either chlorotic or senescent. About 90% of the observed plants showed powdery mildew symptoms. Conidiophores (n = 25) were 74.0 to 160.1 μm × 8.7 to 12.7 μm (on average 120.9 × 10.4 μm) and composed of cylindrical foot cells (length 31.9-72.9 μm, average 50.1 µm) and conidia (mostly 10 conidia) in chains. Barrel-shaped conidia with fibrosin bodies (n = 30) were 12.8 to 21.0 μm × 7.9 to 15 μm, on average 16.7 × 11.3 μm, with a length/width ratio of 1.5. Chasmothecia were not found. Based on these morphologic characteristics, the pathogen was initially identified as Podosphaera macularis (Braun and Cook 2012; Mahaffee et al. 2009). To confirm the identification, two isolates (PDLC0315 and PDLC0412) of P. macularis mycelia and conidia were collected, and mycelia and conidia were combined for a single DNA extraction from each isolate. With the total genomic DNA, the sequences of the internal transcribed spacer (ITS), 5.8S rRNA, the 18S and 28S large subunit ribosomal DNA (LSU) (Bradshaw and Tobin 2020; White et al. 1990), were bi-directionally sequenced and deposited in GenBank (ON862625.1 and ON862630.1). The ON862625.1 and ON862630.1 showed 100% similarity with sequences of P. macularis isolate CT1 (MH687414.1). Phylogenetic analyses based on the combined ITS and 28S rDNA sequences indicated that the two specimens, PDLC0315 and PDLC0412 formed a monophyletic clade together with sequences retrieved from Podosphaera macularis CT1 and Head quarter 31 (KX842348.1). The pathogenicity test with the fungus was confirmed by gently pressing the infected leaves onto three healthy wild plants from the same geographical location. Three uninoculated wild plants served as controls. Six inoculated and non-inoculated plants were placed in different growth chambers with a 16-h photoperiod at 22±2°C and 70% of relative humidity. After 10 to 14 days, powdery mildew colonies developed on inoculated plants. Non-inoculated control plants did not show any symptoms. The fungus on inoculated leaves was morphologically identical to that first observed in the garden. As far as we know, this study is the first report of powdery mildew disease in Humulus scandens caused by Podosphaera macularis in China. Rapid expansion and wild distribution of H. scandens could lead to increased powdery mildew risk in outdoor cultivation. Due to the invasive potential of the powdery mildew fungi, this record is important in the context of the range extension of Podosphaera macularis.

Rapid detection of pyrethroid resistance‐associated mutation L1014F in the field based on loop‐mediated isothermal amplification technique

AbstractMyzus persicae (Sulzer) is a globally distributed crop pest with a wide host range. The L1014F mutation in the voltage‐gated sodium channel has been implicated in mediating resistance to pyrethroid insecticides in M. persicae. Control failures in crop production are frequently attributed to inaccurate or delayed detection of M. persicae resistance. Thus, simple and effective tools for detecting resistant M. persicae are required to develop rapid and flexible pest control strategies. The resistance assay and L1014F mutation frequency assay results showed that the L1014F mutation frequency could infer M. persicae's resistance level to pyrethroid insecticides. Therefore, in our study, we established a Loop‐Mediated Isothermal Amplification (LAMP)‐based method to detect the L1014F mutation in the voltage‐gated sodium channel of M. persicae. This method uses single aphid crude extract DNA as a template, and the full reaction can be completed in 1 h in a relaxed temperature range. The results could also be obtained by colour change without any molecular detection. We have also optimized the reaction system and procedures to improve the accuracy of the assay to over 95%. Based on this, we can detect the mutation frequency of voltage‐gated sodium channel L014F in M. persicae field populations in a non‐laboratory setting, such as in the field, in a stable, accurate, and rapid manner. This method facilitates quick determination of the resistance of M. persicae populations, followed by guide field control.

P-713 Preimplantation DNA methylation screening (PIMS) illustrates the outcome of assisted reproductive technology

Abstract Study question It is unknown whether using DNA methylation can improve the outcome of ART and can exclude the imprinted gene diseases. Summary answer Our study provides a criterion on selecting blastocyst according to DNA methylome, which can improve live birth rate and decrease birth defect rate. What is known already How to select the embryos is one key factor that can determine the successful rate of ART. As aneuploidy exists in 20-30% of blastocysts, preimplantation genetic screening (PGS) has been widely used, which increases the live birth rate. Unfortunately, the live birth rate remains around 50% even with the help of PGS. DNA methylation is known to play important function during embryogenesis. Previous study has shown that a large proportion of human embryos have abnormal DNA methylome. However, whether DNA methylation pattern can affect the clinical outcome of ART has not been investigated in clinics. Study design, size, duration To determine whether PIMS method can increase live birth rate during ART, the whole genome DNA methylation of 3-5 biopsied cells from trophectoderm were measured. The methylation level and chromosome copy number variation (CNV) were analyzed by using the methylome data. elective single embryo transfer (eSET) will be performed for embryos with euploid chromosome. The live birth rate, pregnant rate and abortion rate will be examined. 182 families including 800 blastocysts were enrolled in PIMT. Participants/materials, setting, methods Eligible couples include advanced maternal age, recurrent pregnancy loss with unknown reason, recurrent implantation failure with unknown reason, severe oligoteratozoospermia. Eligible couples must have two or more good-quality blastocysts. We call a blastocyst as good quality if the blastocyst is 4BC or better according to the Gardner morphologic criteria on day 5-6 of embryo culture. Main results and the role of chance No one has ever performed clinical trial to investigate the impact of DNA methylation of blastocyst on the outcome of artificial assisted reproductive (ART). Here, we performed PIMS using the biopsied trophectoderm of 800 blastocysts. Chromosome copy number variation and DNA methylation pattern were analyzed by using methylome data. 162 of euploid embryos were transferred. Our data show that significant variation of DNA methylation level of blastocysts can be observed in both younger women (£37 years old) and older women (>37 years). Blastocysts with global methylation level around 0.26 have the highest birth and pregnancy rate, but the lowest abortion rate. The higher the difference methylation value from 0.26 is, the lower the birth rate becomes. Furthermore, by using artificial intelligence method according to different methylated regions between the embryos succeeding in live-birth and those failing in live-birth, we can robustly predict the clinical outcome of blastocysts. PIMS method can find the abnormality of DNA methylation states in the imprinted control regions which can cause the imprinted diseases. Our study provides a criterion on selecting blastocyst according to DNA methylome during ART, which can potentially improve the live birth rate and decrease the birth defect rate. Limitations, reasons for caution Because of the low coverage of single cell DNA methylome, PIMS method cannot cover all imprinted control regions. Wider implications of the findings PIMS method can potentially find more human diseases which is caused by DNA methylation mutation. Trial registration number NCT03642574

P-053 Sperm aneuploidy and DNA integrity pre- and post-chemotherapy in men with testicular cancer

Abstract Study question Are fragmentation levels (single-double stranded DNA breaks) or aneuploidy frequencies in sperm increased in patients with testicular cancer who recovered spermatogenesis after chemotherapy (CT)? Summary answer Similar levels of sperm DNA fragmentation and sperm aneuploidy frequency are found in men with testicular cancer pre- and post-chemotherapy. What is known already Testicular cancer is the most frequent solid tumor among young men. Therefore, the restoration of fertility and achievement of fatherhood in survivors have become important concerns. The integrity of genetic material in sperm is crucial since some paternal genes are essential for early embryo development. Although previous studies analyzed the effect of CT on sperm DNA integrity, they did not differentiate single vs. double-stranded DNA breaks. Few data are available on aneuploidy in sperm from testicular cancer survivors, so it remains to be elucidated whether cancer treatments may lead to an increased risk of aneuploidy syndromes in offspring. Study design, size, duration Prospective multicenter study including 31 males with a history of testicular cancer who requested semen cryopreservation between 2007-2021. All patients included underwent CT and recovered spermatogenesis once treatment ended. Post-CT samples were collected and seminal parameters, single-double stranded DNA breaks and the proportion of sperm aneuploidies were compared with one aliquot of the initial ejaculate. The project was approved by an Institutional Review Board and written informed consent for participation was obtained from each subject. Participants/materials, setting, methods Patient´s age prior CT was 31.23yrs (IC 95%: 29.29-33.17). Mean interval time between 1st and 2nd samples was 4.53yrs (IC 95%: 3.42-5.64). TUNEL quantified overall sperm DNA fragmentation by flow cytometry analyzing at least 20,000 cells, while γH2AX staining was applied for double DNA fragmentation assessment. Sperm aneuploidies were calculated by using fluorescence in situ hybridization (FISH) probes for chromosomes 13, 18, 21, X, Y. A paired t-test was used to compare variables pre- and post-CT. Main results and the role of chance Similar results were found when sperm quality parameters were compared. Mean sperm concentration pre-CT was 33.03M/mL (IC 95%: 23.98-42.07) and 36.41 M/mL (IC 95%: 27.85-44.96) in post-CT ejaculates (N.S.). A comparable rate of motile spermatozoa was observed in pre-CT vs. post-CT samples [(43.81% (IC 95%: 38.42-49.19) vs. 44.73% (IC 95%: 38.95-50.51), N.S.]. Regarding TUNEL assay results, the sperm DNA fragmentation rate was 21.65% (IC 95%: 17.66-25.63) in pre-CT samples and 23.00% (IC 95%: 19.90-26.61) in post-CT samples (N.S.). Furthermore, similar results were obtained when double-stranded fragmentation levels (H2AX) were compared between pre and post-CT [10.2% (IC 95%: 6.45-13.96) vs. 12.44% (IC 95%: 8.75-16.13) N.S.]. Comparable sperm disomic proportions were found in PRE and POST-CT samples: [(Cr.13 =0.02%, Cr.18 =0.01%, Cr.21=0.30%, Cr.XY=0.84%) vs. (Cr.13 =0%, Cr.18 =0%, Cr.21=0%, Cr.XY=0.33%), N.S]. Results were also evaluated according to the time elapsed between the PRE and POST -CT samples. No significant differences were found for seminal values (sperm concentration, p = 0.26; motility, p = 0.68), overall sperm DNA fragmentation (p = 0.68), double-strand DNA fragmentation (p = 0.90) and the frequency of sperm disomies (Cr. 13 p = 0.79, Cr.18 p = 0.46, Cr. 21 p = 0.85, Cr.XY p = 0.05). Limitations, reasons for caution The main limitations of the present study are that we only recruited patients who recovered spermatogenesis and that our data evaluated a long-term effect of CT. Wider implications of the findings All men wishing to have their own children after gonadotoxic treatment should receive adequate counseling about the side effects of the therapy. Based on our results on sperm DNA integrity and euploidy, patients who recovered spermatogenesis should not be advised to use samples that were preserved before undergoing CT. Trial registration number na

Deciphering molecular mechanisms of phase separation in RNA biology by single-molecule biophysical technologies.

Ribonucleic acid (RNA) biology has emerged as one of the most important areas in modern biology and biomedicine. RNA and RNA-binding proteins (RBPs) are involved in forming biomolecular condensates, which are crucial for RNA metabolism. To quantitively decipher the molecular mechanisms of RNP granules, researchers have turned to single-molecule biophysical techniques, such as single-molecule Förster resonance energy transfer (smFRET), in vivo single-molecule imaging technique with single particle tracking (SPT), DNA Curtains, optical tweezers, and atomic force microscopy (AFM). These methods are used to investigate the molecular biophysical properties within RNP granules, as well as the molecular interactions between RNA and RBPs and RBPs themselves, which are challenging to study using traditional experimental methods of the liquid-liquid phase separation (LLPS) field, such as fluorescence recovery after photobleaching (FRAP). In this work, we summarize the applications of single-molecule biophysical techniques in RNP granule studies and highlight how these methods can be used to reveal the molecular mechanisms of RNP granules.

Computational analysis on the dissemination of non-B DNA structural motifs in promoter regions of 1180 cellular genomes

The promoter regions of gene regulation are under evolutionary constraints and earlier studies uncovered that they are characterized by enrichment of functional non-B DNA structural signatures like curved DNA, cruciform DNA, G-quadruplex, triple-helical DNA, slipped DNA structures, and Z-DNA. However, these studies are restricted to a few model organisms, single non-B DNA motif types, or whole genomic sequences, and their comparative accumulation in promoter regions of different domains of life has not been reported comprehensively. In this study, for the first time, we investigated the preponderance of non-B DNA-prone motifs in promoter regions in 1180 genomes belonging to 28 taxonomic groups using the non-B DNA Motif Search Tool (nBMST). The trends suggest that they are predominant in promoters compared to the upstream and downstream regions of all three domains of life and variably linked to taxonomic groups. Cruciform DNA motif is the most abundant form of non-B DNA, spanning from archaea to lower eukaryotes. Curved DNA motifs are prominent in host-associated bacteria, and suppressed in mammals. Triplex-DNA and slipped DNA structure repeats are discretely dispersed in all lineages. G-quadruplex motifs are significantly enriched in mammals. We also observed that the unique enrichment of non-B DNA in promoters is strongly linked to genome GC, size, evolutionary time divergence, and ecological adaptations. Overall, our work systematically reports the unique non-B DNA structural landscape of cellular organisms from the perspective of the cis-regulatory code of genomes.

Episomal and chromosomal DNA replication and recombination in Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebiasis. DNA replication studies in E. histolytica first started with the ribosomal RNA genes located on episomal circles. Unlike most plasmids, Entamoeba histolytica rDNA circles lacked a fixed origin. Replication initiated from multiple sites on the episome, and these were preferentially used under different growth conditions. In synchronized cells the early origins mapped within the rDNA transcription unit, while at later times an origin in the promoter-proximal upstream intergenic spacer was activated. This is reminiscent of eukaryotic chromosomal replication where multiple potential origins are used. Biochemical studies on replication and recombination proteins in Entamoeba histolytica picked up momentum once the genome sequence was available. Sequence search revealed homologs of DNA replication and recombination proteins, including meiotic genes. The replicative DNA polymerases identified included the α, δ, ε of polymerase family B; lesion repair polymerases Rev1 and Rev3; a translesion repair polymerase of family A, and five families of polymerases related to family B2. Biochemical analysis of EhDNApolA confirmed its polymerase activity with expected kinetic constants. It could perform strand displacement, and translesion synthesis. The purified EhDNApolB2 had polymerase and exonuclease activities, and could efficiently bypass some types of DNA lesions. The single DNA ligase (EhDNAligI) was similar to eukaryotic DNA ligase I. It was a high-fidelity DNA ligase, likely involved in both replication and repair. Its interaction with EhPCNA was also demonstrated. The recombination-related proteins biochemically characterized were EhRad51 and EhDmc1. Both shared the canonical properties of a recombinase and could catalyse strand exchange over long DNA stretches. Presence of Dmc1 indicates the likelihood of meiosis in this parasite. Direct evidence of recombination in Entamoeba histolytica was provided by use of inverted repeat sequences located on plasmids or chromosomes. In response to a variety of stress conditions, and during encystation in Entamoeba invadens, recombination-related genes were upregulated and homologous recombination was enhanced. These data suggest that homologous recombination could have critical roles in trophozoite growth and stage conversion. Availability of biochemically characterized replication and recombination proteins is an important resource for exploration of novel anti-amoebic drug targets.

Single-molecule characterization of target search dynamics of DNA-binding proteins in DNA-condensed droplets.

Target search models of DNA-binding proteins in cells typically consider search mechanisms that include 3D diffusion and 1D sliding, which can be characterized by single-molecule tracking on DNA. However, the finding of liquid droplets of DNA and nuclear components in cells cast doubt on extrapolation from the behavior in ideal non-condensed DNA conditions to those in cells. In this study, we investigate the target search behavior of DNA-binding proteins in reconstituted DNA-condensed droplets using single-molecule fluorescence microscopy. To mimic nuclear condensates, we reconstituted DNA-condensed droplets using dextran and PEG polymers. In the DNA-condensed droplets, we measured the translational movement of four DNA-binding proteins (p53, Nhp6A, Fisand Cas9) and p53 mutants possessing different structures, sizes, and oligomeric states. Our results demonstrate the presence of fast and slow mobility modes in DNA-condensed droplets for the four DNA-binding proteins. The slow mobility mode capability is correlated strongly to the molecular size and the number of DNA-binding domains on DNA-binding proteins, but only moderately to the affinity to single DNA segments in non-condensed conditions. The slow mobility mode in DNA-condensed droplets is interpreted as a multivalent interaction mode of the DNA-binding protein to multiple DNA segments.

A Single DNA Point Mutation Leads to the Formation of a Cysteine-Tyrosine Crosslink in the Cysteine Dioxygenase from Bacillus subtilis.

Cysteine dioxygenase (CDO) is a non-heme iron-containing enzyme that catalyzes the oxidation of cysteine (Cys) to cysteine sulfinic acid (CSA). Crystal structures of eukaryotic CDOs revealed the presence of an unusual crosslink between the sulfur of a cysteine residue (C93 in Mus musculus CDO, MmCDO) and a carbon atom adjacent to the phenyl group of a tyrosine residue (Y157). Formation of this crosslink occurs over time as a byproduct of catalysis and increases the catalytic efficiency of CDO by at least 10-fold. Interestingly, in bacterial CDOs, the residue corresponding to C93 is replaced by a highly conserved glycine (G82 in Bacillus subtilis CDO, BsCDO), which precludes the formation of a C-Y crosslink in these enzymes; yet bacterial CDOs achieve turnover rates paralleling those of fully crosslinked eukaryotic CDOs. In the present study, we prepared the G82C variant of BsCDO to determine if a single DNA point mutation could lead to C-Y crosslink formation in this enzyme. We used gel electrophoresis, peptide mass spectrometry, electron paramagnetic resonance spectroscopy, and kinetic assays to characterize this variant alongside the natively crosslinked wild-type (WT) MmCDO and the natively non-crosslinked WT BsCDO. Collectively, our results provide compelling evidence that the G82C BsCDO variant is indeed capable of C-Y crosslink formation. Our kinetic studies indicate that G82C BsCDO has a reduced catalytic efficiency compared to WT BsCDO and that activity increases as the ratio of crosslinked to non-crosslinked enzyme increases. Finally, by carrying out a bioinformatic analysis of the CDO family, we were able to identify a large number of putatively crosslinked bacterial CDOs, the majority of which are from Gram-negative pathogenic bacteria.

Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore.

This paper describes a method for the real-time counting and extraction of DNA molecules at the single-molecule level by nanopore technology. As a powerful tool for electrochemical single-molecule detection, nanopore technology eliminates the need for labeling or partitioning sample solutions at the femtoliter level. Here, we attempt to develop a DNA filtering system utilizing an α-hemolysin (αHL) nanopore. This system comprises two droplets, one filling with and one emptying DNA molecules, separated by a planar lipid bilayer containing αHL nanopores. The translocation of DNA through the nanopores is observed by measuring the channel current, and the number of translocated molecules can also be verified by quantitative polymerase chain reaction (qPCR). However, we found that the issue of contamination seems to be an almost insolvable problem in single-molecule counting. To tackle this problem, we tried to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method. Although further efforts are still needed to achieve a single-molecule filter with electrical counting, our proposed method shows a linear relationship between the electrical counting and qPCR estimation of the number of DNA molecules.

The LASSIE MPS panel: Predicting externally visible traits in dogs for forensic purposes

Predicting the outward appearance of dogs via their DNA, also known as Canine DNA Phenotyping, is a young, emerging field of research in forensic genetics. The few previous studies published in this respect were restricted to the consecutive analysis of single DNA markers, a process that is time- and sample-consuming and therefore not a viable option for limited forensic specimens. Here, we report on the development and evaluation of a Massively Parallel Sequencing (MPS) based molecular genetic assay, the LASSIE MPS Panel. This panel aims to predict externally visible as well as skeletal traits, which include coat color, coat pattern, coat structure, tail morphology, skull shape, ear shape, eye color and body size from DNA using 44 genetic markers in a single molecular genetic assay. A biostatistical naïve Bayes classification approach was applied to identify the most informative marker combinations for predicting phenotypes. Overall, the predictive performance was characterized by a very high classification success for some of the trait categories, and high to moderate success for others. The performance of the developed predictive framework was further evaluated using blind samples from three randomly selected dog individuals, whose appearance was well predicted.