Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

Functional analysis of the new barley gene HvKu80 indicates that it plays a key role in double-strand DNA break repair and telomere length regulation.

Genotoxic stress causes a reduced stability of the plant genome and has a detrimental effect on plant growth and productivity. Double-strand breaks (DSBs) are the most harmful of all DNA lesions because they cause the loss of genetic information on both strands of the DNA helix. In the presented study the coding and genomic sequences of the HvKu80 gene were determined. A mutational analysis of two fragments of HvKu80 using TILLING (Targeting Induced Local Lesions IN Genomes) allowed 12 mutations to be detected, which resulted in identification of 11 alleles. Multidirectional analyses demonstrated that the HvKu80 gene is involved in the elimination of DSBs in Hordeum vulgare. The barley mutants carrying the identified ku80.c and ku80.j alleles accumulated bleomycin-induced DSBs to a much greater extent than the parent cultivar 'Sebastian'. The altered reaction of the mutants to DSB-inducing agent and the kinetics of DNA repair in these genotypes are associated with a lower expression level of the mutated gene. The study also demonstrated the significant role of the HvKu80 gene in the regulation of telomere length in barley.

Single-copy detection of somatic variants from solid and liquid biopsy.

Accurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

Examination of oral biofilm microbiota in patients using fixed orthodontic appliances in order to prevent risk factors for health complications.

INTRODUCTION AND OBJECTIVE: In recent decades the use of orthodontic appliances in Poland has increased; however, data on their influence on changes of components of the microbiome connected with oral biofilm are scarce. The objective of this study was to evaluate oral microbiota in terms of their role as risk factors for health complications. MATERIAL AND METHODS: The study included 100 patients treated with removable or fixed appliances. Oral hygiene and gingival health were determined, and periodontal swabs taken from each patient for parasitological, bacteriological and mycological microscopic and in vitro examinations. RESULTS: Oral protists and various pathogenic and opportunistic bacterial and fungal strains were identified in the superficial layer of biofilm. A higher prevalence of bacteria, Enterococcus faecalis, E. faecium, Staphylococcus aureus and Escherichia coli, and various strains of yeast-like fungi from the Candida albicans group, occurred in patients treated with the fixed appliance than in those using a removable appliance or not treated orthodontically. In some periodontal samples from patients treated with fixed appliances, cysts of the Acanthamoeba spp. were found. CONCLUSIONS: The use of orthodontic appliances alters the status of the oral cavity; it has impact on the colonization of oral biofilm by opportunistic/pathogenic strains, and increases the risk of their dissemination to various human tissues and organs. Pretreatment examination of oral microbiome, its monitoring particularly during treatment with fixed appliances, and preventive elimination of the potentially pathogenic strains to avoid health complications, are highly recommended, especially in patients with impaired immunity.

HorTILLUS-A Rich and Renewable Source of Induced Mutations for Forward/Reverse Genetics and Pre-breeding Programs in Barley (Hordeum vulgare L.).

TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS ( Hordeum-TILLING-University of Silesia) population created for spring barley cultivar "Sebastian" after double-treatment of seeds with two chemical mutagens: sodium azide (NaN3) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M2 plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M3 progeny of 3,481 M2 individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072-6,912 M2 plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.

[Thermographic assessment of thermal effects of Er:YAG laser in periodontal surgery]. / Termograficzna ocena ciepinego oddzialywania lasera Er:YAG w chirurgii periodontologicznej.

AIM OF THE STUDY: An assessment of thermal effect of Er:YAG laser (KEYII, KaVo) on oral soft tissues in select procedures. MATERIAL AND METHODS: Experimental researches were carried out on Wistar rats. To measure the temperature changes the thermal imaging camera (ThermaCAM SC3000, FLIR Systems) was used. RESULTS: There has been a significant increase of temperature observed on the end of optical fibre: the mean temperature ranged from 270 to 360 degrees C (at laser energy of 100 mJ and repetition rate of 25 Hz) and from 230 to 290 degrees C (300 mJ, 15 Hz). On the surface of oral mucosa thermal changes at the time of laser frenulectomy was analysed along the line of incision. The temperature above 50 degrees C was recorded on the length of 2 mm (at 100 mJ) and 3 mm (at 300 mJ). The temperature maintained on this level for about 0.4 s. On the surface of tongue during lingual mucosa excision the temperature above 40 degrees C was observed on the length of 1.6 mm (80 mJ, 2 Hz) or 2.5 mm (160 mJ, 2 Hz). The rate of cooling for both cases was lower than 0.5 s. CONCLUSION: To prevent undesirable thermal side effects from an Er:YAG laser optical fibre should be moved very fluently in non-contact mode.

Alleles of newly identified barley gene HvPARP3 exhibit changes in efficiency of DNA repair.

Genome integrity is constantly challenged by endo- and exogenous DNA-damaging factors. The influence of genotoxic agents causes an accumulation of DNA lesions, which if not repaired, become mutations that can cause various abnormalities in a cell metabolism. The main pathway of DSB repair, which is based on non-homologous recombination, is canonical non-homologous end joining (C-NHEJ). It has been shown that this mechanism is highly conserved in both Pro- and Eukaryotes. The mechanisms that underlie DSB repair through C-NHEJ have mainly been investigated in mammalian systems, and therefore our knowledge about this process is much more limited as far as plants, and crop plants in particular, are concerned. Recent studies have demonstrated that PARP3 is an important response factor to the presence of DSB in a genome. The aims of this study were to identify the sequence of the barley PARP3 gene, to perform a mutational analysis of the sequence that was identified using the TILLING (Targeting Induced Local Lesions IN Genomes) method and to phenotype the mutants that were identified through their exposure to mutagenic treatment with the DSB-inducing chemical--bleomycin. A functional analysis led to the identification of a series of parp3 alleles. The mutants were characterized using several different approaches, including quantifying the DSB and γH2AX foci, which validated the function of the HvPARP3 gene in DSB repair in barley. The potential involvement of the HvPARP3 gene in the regulation of telomere length in barley was also analyzed.