Nucleotide-selective amplification and array-based detection for identifying multiple somatic mutations.

In the context of personalized and cost-effective treatment, knowledge of the mutational status of specific genes is advantageous to predict which patients are responsive to therapies. As an alternative to one-by-one detection or massive sequencing, the presented genotyping tool determines multiple polymorphic sequences that vary a single nucleotide. The biosensing method includes an effective enrichment of mutant variants and selective recognition by colorimetric DNA arrays. The proposed approach is the hybridization between sequence-tailored probes and products from PCR with SuperSelective primers to discriminate specific variants in a single locus. A fluorescence scanner, a documental scanner, or a smartphone captured the chip images to obtain spot intensities. Hence, specific recognition patterns identified any single-nucleotide change in the wild-type sequence overcoming qPCR methods and other array-based approaches. Studied mutational analyses applied to human cell lines provided high discrimination factors, the precision was 95%, and the sensitivity was 1% mutant of total DNA. Also, the methods showed a selective genotyping of the KRAS gene from tumorous samples (tissue and liquid biopsy), corroborating results by NGS. The developed technology supported on low-cost robust chips and optical reading provides an attractive pathway toward implementing fast, cheap, reproducible discrimination of oncological patients.

Description of Copy Number Variations in a Series of Children and Adolescents with FASD in Reunion Island.

BACKGROUND: Fetal Alcohol Spectrum Disorders (FASD) are the most common cause of neurocognitive impairment and social inadaptation, affecting 1 birth in 100. Despite the existence of precise diagnostic criteria, the diagnosis remains difficult, often confounded with other genetic syndromes or neurodevelopmental disorders. Since 2016, Reunion Island has been a pilot region for the identification, diagnosis, and care of FASD in France. OBJECTIVE: To evaluate the prevalence and the types of Copy Number Variations (CNV) in FASD patients. METHODS: A retrospective chart review of 101 patients diagnosed with FASD in the Reference Center for developmental anomalies and in the FASD Diagnostic Center of the University Hospital was performed. Records of all patients were reviewed to obtain their medical history, family history, clinical phenotype, and investigations, including genetic testing (CGH- or SNP-array). RESULTS: A rate of 20.8% (n = 21) of CNVs was found including 57% (12/21) of pathogenic variants and 29% (6/21) of variants of uncertain signification (VUS). CONCLUSION: A particularly high number of CNVs was found in children and adolescents with FASD. It reinforces the plea for a multidisciplinary approach for developmental disorders to explore both environmental factors, such as avoidable teratogens and intrinsic vulnerabilities, especially genetic determinants.

Application potential of chicken DNA chip in domestic pigeon species - Preliminary results.

Introducing the SNP technology to pigeon breeding will enhance the competitiveness of a sector that produces one of the healthiest and best quality meats. The present study aimed to test the applicability of the Illumina Chicken_50K_CobbCons array on 24 domestic pigeon individuals from the Mirthys hybrids and Racing pigeon breeds. A total of 53,313 SNPs were genotyped. Principal component analysis shows a significant overlap between the two groups. The chip performed poorly in this data set, with a call rate per sample of 0.474 (49%). The low call rate was likely due to an increase in the evolutionary distance. A total of 356 SNPs were retained after a relatively strict quality control. We have demonstrated that it is technically feasible to use a chicken microarray chip on pigeon samples. Presumably, with a larger sample size and by assigning phenotypic data, efficiency would be improved, allowing more thorough analyses, such as genome-wide association studies.

Comparison of the performance of HPV DNA chip test and HPV PCR test in cervical cancer screening in rural China.

Background: This study aimed to evaluate the performance of two different principles of HPV testing in primary cervical cancer screening and ASC-US triage in rural areas. Methods: 3,328 and 3,913 women were enrolled in Shanxi, China in 2017 and 2018, respectively, and screened using liquid-based cytology and different HPV tests with a 4-year follow-up. Different screening methods commonly used in clinical practice were evaluated. Results: In the HPV PCR test cohort, the prevalence of HPV infection was 14.90%. A total of 38 cases of CIN2+ were identified at baseline, 2 of which were in the HPV-negative cohort and the rest in the HPV-positive cohort (2 = 186.85, p < 0.001). Fifty-three cases of CIN2+ were accumulated over 4 years. The HPV infection rate in the HPV DNA chip test cohort was 21.10%. A total of 26 CIN2+ cases were identified at baseline, all in the HPV-positive population (2 = 92.96, p < 0.001). 54 CIN2+ cases were cumulative over 4 years. At 4-year follow-up, HPV-negative results were significantly more protective against cervical intraepithelial neoplasia grade 2 or worse (CIN2+) than normal cytologic results at baseline. HPV screening was more sensitive and specific than cytologic screening (using ASC-US as the threshold) and performed better on the HPV DNA microarray test. In addition, compared with HPV 16/18 testing, sensitivity increases and specificity decreases when using HPV testing for cytologic ASC-US triage, regardless of which HPV test is used. Conclusion: In the rural areas where we implemented the study, HPV tests performed well for screening than LBC and HPV DNA chip testing performed better than HPV PCR testing in the screening cohort. Optimal screening was achieved technically when used in combination with LBC for ASC-US population triage, without thinking the feasibility for resource availability.

Cell-Free Gene Expression from DNA Brushes.

Linear double-stranded DNA polymers coding for synthetic genes immobilized on a surface form a brush as a center for cell-free gene expression, with DNA density 102-103 fold higher than in bulk solution reactions. A brush localizes the transcription-translation machinery in cell extracts or in cell-free reconstituted reactions from purified components, creating a concentrated source of RNA and proteins. Newly synthesized molecules can form circuits regulating gene expression in the same brush or adjacent ones. They can also assemble into functional complexes and machines such as ribosomal units, then analyzed by capture on prepatterned antibodies or by cascaded reactions. DNA brushes are arranged as a single center or multiple ones on a glass coverslip, in miniaturized compartments carved in silicon wafers, or in elastomeric microfluidic devices. Brushes create genetically programmable artificial cells with steady-state dynamics of protein synthesis. Here, we provide the basic procedure for surface patterning, DNA immobilization, capture of protein products on antibody traps and fluorescent imaging. The method of DNA brush surface patterning enables simple parallelization of cell-free gene expression reactions for high throughput studies with increased imaging sensitivity.

DNA Methylation Differences in Peripheral Blood of Patients with Anaphylaxis.

ABSTRACT: Objective To study the DNA methylation of nucleated cells in peripheral blood of patients died from anaphylactic shock caused by cephalosporin drugs and to provide a new research direction and basis for the forensic diagnosis of shock caused by drug hypersensitiveness. Methods Methylation microarray was used to detect DNA methylation of nucleated cells in peripheral blood of patients died from anaphylactic shock caused by cephalosporin drugs and normal subjects. Sequencing data and chip data were analyzed for differences in DNA methylation using R language methylkit, ChAMP package. Random forest algorithm was used to evaluate the importance of the DNA methylation differential sites. Results Differential sites of DNA methylation highly associated with anaphylaxis caused by cephalosporin drugs were obtained at loci such as ETS1, PRR23B and GNAS. Conclusion Cephalosporin allergy is associated with DNA methylation, and DNA methylation may be a new strategy for forensic identification of anaphylactic shock and death.

[The current status and future prospects of DNA computing].

As the demand for high-performance computing continues to grow, traditional computing models are facing unprecedented challenges. Among the many emerging computing technologies, DNA computing has attracted much attention due to its low energy consumption and parallelism. The DNA circuit, which is the basis for DNA computing, is an important technology for the regulation and processing of the molecular information. This review highlights the basic principles of DNA computing, summarizes the latest research progress, and concludes with a discussion of the challenges of DNA computing. Such integrated molecular computing systems are expected to be widely used in the fields of aerospace, information security and defense system.

A Label-Free Diamond Microfluidic DNA Sensor Based on Active Nitrogen-Vacancy Center Charge State Control.

We propose a label-free biosensor concept based on the charge state manipulation of nitrogen-vacancy (NV) quantum color centers in diamond, combined with an electrochemical microfluidic flow cell sensor, constructed on boron-doped diamond. This device can be set at a defined electrochemical potential, locking onto the particular chemical reaction, whilst the NV center provides the sensing function. The NV charge state occupation is initially prepared by applying a bias voltage on a gate electrode and then subsequently altered by exposure to detected charged molecules. We demonstrate the functionality of the device by performing label-free optical detection of DNA molecules. In this experiment, a monolayer of strongly cationic charged polymer polyethylenimine is used to shift the charge state of near surface NV centers from negatively charged NV- to neutral NV0 or dark positively charged NV+. Immobilization of negatively charged DNA molecules on the surface of the sensor restores the NV centers charge state back to the negatively charged NV-, which is detected using confocal photoluminescence microscopy. Biochemical reactions in the microfluidic channel are characterized by electrochemical impedance spectroscopy. The use of the developed electrochemical device can also be extended to nuclear magnetic resonance spin sensing.

Evaluation of human papillomavirus (HPV) genotyping assays using type-specific HPV L1 reference DNA.

BACKGROUND: Human papillomaviruses (HPV) are known to play a central etiological role in the development of cervical cancer. General HPV genotyping methods consist of PCR with consensus primers combined with various detection methods. OBJECTIVE: The aim was to develop HPV L1 DNA reference materials to evaluate the sensitivity, specificity, and accuracy of genotyping results obtained from the HPV DNA Genotyping Chip (HPV CHIP) and RFMP assays. METHODS: In this study, the Ministry of Food and Drug Safety (MFDS) established reference DNA materials for the L1 gene from 41 subtypes of anogenital HPV to aid in genotyping human papillomavirus (HPV) strains. Of these, 22 subtypes were obtained from cervical scrape samples of Korean women and 19 subtypes were synthesized. These reference materials include 13 high-risk types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), 3 probable high-risk types (HPV-26, 53, and 66), 16 low-risk types (HPV-6, 10, 11, 27, 34, 40, 42, 43, 44, 54, 55, 61, 70, 72, 73, and 81), and 8 undetermined-risk types (HPV-3, 57, 62, 67, 69, 71, 74, and 84). After confirming the sequences by standard methods, these HPV L1 DNA reference materials were then used to compare results from the HPV DNA Genotyping Chip (HPV CHIP) and restriction fragment mass polymorphism (RFMP) assays. RESULTS: Data collected from the HPV CHIP and RFMP assay showed comparably high sensitivity and accuracy. Both assays could detect 102 or more copies/µl of HPV L1 DNA from 39 types of HPV, with higher accuracy in detecting samples with mixed types of HPV. CONCLUSION: The present study confirms the HPV L1 DNA reference materials developed by MFDS are reliable and useful for the evaluation of HPV genotyping assays.

The current status and future prospects of DNA computing / 生物工程学报

As the demand for high-performance computing continues to grow, traditional computing models are facing unprecedented challenges. Among the many emerging computing technologies, DNA computing has attracted much attention due to its low energy consumption and parallelism. The DNA circuit, which is the basis for DNA computing, is an important technology for the regulation and processing of the molecular information. This review highlights the basic principles of DNA computing, summarizes the latest research progress, and concludes with a discussion of the challenges of DNA computing. Such integrated molecular computing systems are expected to be widely used in the fields of aerospace, information security and defense system.

DNA Methylation Differences in Peripheral Blood of Patients with Anaphylaxis / 法医学杂志

Objective To study the DNA methylation of nucleated cells in peripheral blood of patients died from anaphylactic shock caused by cephalosporin drugs and to provide a new research direction and basis for the forensic diagnosis of shock caused by drug hypersensitiveness. Methods Methylation microarray was used to detect DNA methylation of nucleated cells in peripheral blood of patients died from anaphylactic shock caused by cephalosporin drugs and normal subjects. Sequencing data and chip data were analyzed for differences in DNA methylation using R language methylkit, ChAMP package. Random forest algorithm was used to evaluate the importance of the DNA methylation differential sites. Results Differential sites of DNA methylation highly associated with anaphylaxis caused by cephalosporin drugs were obtained at loci such as ETS1, PRR23B and GNAS. Conclusion Cephalosporin allergy is associated with DNA methylation, and DNA methylation may be a new strategy for forensic identification of anaphylactic shock and death.

Recurrent Drought Conditions Enhance the Induction of Drought Stress Memory Genes in Glycine max L.

Plants remember what they have experienced and are thereby able to confront repeated stresses more promptly and strongly. A subset of the drought responsive genes, called stress memory genes, displayed greatly elevated levels under recurrent drought conditions. To screen for a set of drought stress memory genes in soybean (Glycine max L.), we designed a 180K DNA chip comprising 60-bp probes synthesized in situ to examine 55,589 loci. Through microarray analysis using the DNA chip, we identified 2,162 and 2,385 genes with more than fourfold increases or decreases in transcript levels, respectively, under initial (first) drought stress conditions, when compared with the non-treated control. The transcript levels of the drought-responsive genes returned to basal levels during recovery (watered) states, and 392 and 613 genes displayed more than fourfold elevated or reduced levels, respectively, under subsequent (second) drought conditions, when compared to those observed under the first drought stress conditions. Gene Ontology and MapMan analyses classified the drought-induced memory genes exhibiting elevated levels of transcripts into several functional categories, including those involved in tolerance responses to abiotic stresses, which encode transcription factors, protein phosphatase 2Cs, and late embryogenesis abundant proteins. The drought-repressed memory genes exhibiting reduced levels of transcripts were classified into categories including photosynthesis and primary metabolism. Co-expression network analysis revealed that the soybean drought-induced and -repressed memory genes were equivalent to 172 and 311 Arabidopsis genes, respectively. The soybean drought stress memory genes include genes involved in the dehydration memory responses of Arabidopsis.

Evidence of distinct gene functional patterns in GC-poor and GC-rich isochores in Bos taurus.

Vertebrate genomes are mosaics of megabase-size DNA segments with a fairly homogeneous base composition, called isochores. They are divided into five families characterized by different guanine-cytosine (GC) levels and linked to several functional and structural properties. The increased availability of fully sequenced genomes allows the investigation of isochores in several species, assessing their level of conservation across vertebrate genomes. In this work, we characterized the isochores in Bos taurus using the ARS-UCD1.2 genome version. The comparison of our results with the well-studied human isochores and those of other mammals revealed a large conservation in isochore families, in number, average GC levels and gene density. Exceptions to the established increase in gene density with the increase in isochores (GC%) were observed for the following gene biotypes: tRNA, small nuclear RNA, small nucleolar RNA and pseudogenes that have their maximum number in H2 and H1 isochores. Subsequently, we assessed the ontology of all gene biotypes looking for functional classes that are statistically over- or under-represented in each isochore. Receptor activity and sensory perception pathways were significantly over-represented in L1 and L2 (GC-poor) isochores. This was also validated for the horse genome. Our analysis of housekeeping genes confirmed a preferential localization in GC-rich isochores, as reported in other species. Finally, we assessed the SNP distribution of a bovine high-density SNP chip across the isochores, finding a higher density in the GC-rich families, reflecting a potential bias in the chip, widely used for genetic selection and biodiversity studies.

Rapid detection of deformed wing virus in honeybee using ultra-rapid qPCR and a DNA-chip.

Fast and accurate detection of viral RNA pathogens is important in apiculture. A polymerase chain reaction (PCR)-based detection method has been developed, which is simple, specific, and sensitive. In this study, we rapidly (in 1 min) synthesized cDNA from the RNA of deformed wing virus (DWV)-infected bees (Apis mellifera), and then, within 10 min, amplified the target cDNA by ultra-rapid qPCR. The PCR products were hybridized to a DNA-chip for confirmation of target gene specificity. The results of this study suggest that our method might be a useful tool for detecting DWV, as well as for the diagnosis of RNA virus-mediated diseases on-site.

Design of a user-friendly and rapid DNA microarray assay for the authentication of ten important food fish species.

Seafood is particularly susceptible to the substitution of species. In order to guarantee authentic seafood products, seafood processors and traders must perform self-checks on the authenticity of imported and purchased goods. However, the conventional Sanger sequencing of PCR products for the authentication of seafood species is time-consuming and requires advanced infrastructure. DNA microarrays (DNA chips) with species-specific oligonucleotide probes represent a rapid alternative to sequencing-based species authentication. So far, though, only DNA microarrays for the authentication of land vertebrate species have achieved market success. In this work, a user-friendly DNA microarray assay was developed for the authentication of ten important food fish species that can be performed in four to five hours from start to end. The assay was tested with authenticated specimens from 67 different fish species, and by comparing the probe signal patterns all target species and even closely related non-target species could be distinguished.

Rapid detection of deformed wing virus in honeybee using ultra-rapid qPCR and a DNA-chip

Modulated light-activated electrochemistry at silicon functionalized with metal-organic frameworks towards addressable DNA chips.

Modulated light-activated electrochemistry (MLAE) at semiconductor/liquid interfaces derived from light-addressable potentiometric sensor (LAPS) and light-activated electrochemistry (LAE) for addressable photoelectrochemical sensing has been proposed as a new sensor platform. In this system, a bias voltage is applied to create a depletion layer at the silicon/electrolyte interface. Meanwhile, intensity-modulated light illuminates the movable electrode to generate electron/hole pairs and causes a detectable local AC photocurrent. The AC measurement showed a higher signal-to-noise ratio (SNR) of photocurrents compared to the traditional DC response, while a steeper photocurrent-voltage (I-V) curve than that of LAPS with an insulating layer was obtained. Furthermore, to stabilize and functionalize the silicon substrate, metal-organic framework (MOF) nanoparticles were grown in-situ on the silicon electrode. The successful modification was validated by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The AC photocurrent increased as a result of the adsorption of negatively charged DNA, which contributed to the enhancement of the cathodic reduction process at the semiconductor electrodes, indicating a different response mechanism of MLAE from LAPS. The results obtained demonstrate the potential of MOF functionalized MLAE as a robust platform for light-addressable DNA chips with high sensitivity and specificity.

Simulating DNA Chip Design Using All-Electronic Graphene-Based Substrates.

In this paper, we present a theoretical investigation of an all-electronic biochip based on graphene to detect DNA including a full dynamical treatment for the environment. Our proposed device design is based on the changes in the electronic transport properties of graphene interacting with DNA strands under the effect of the solvent. To investigate these systems, we applied a hybrid methodology, combining quantum and classical mechanics (QM/MM) coupled to non-equilibrium Green's functions, allowing for the calculations of electronic transport. Our results show that the proposed device has high sensitivity towards the presence of DNA, and, combined with the presence of a specific DNA probe in the form of a single-strand, it presents good selectivity towards specific nucleotide sequences.

Application of ultra-rapid qPCR and DNA chips for viral RNA detection and confirmation.

The rapid and accurate detection of the presence of microorganisms, such as viruses, has been an important issue in the fields of public health, as well as agriculture. A PCR-based detection method has been developed and applied in these fields to determine the presence of specific pathogens. Although the major advantage of real-time PCR is the monitoring of amplification and ability to quantify the template genes, the method described here should solve the problem of nonspecific product synthesis. We obtained viral RNA from infected samples by freezing and thawing; we rapidly synthesized cDNA from RNA, and then amplified the cDNA by rapid PCR in 10 Min. Finally, the PCR products were hybridized and quickly confirmed to be the target analyte on a DNA chip. Our newly proposed methods overcome the drawbacks of PCR-based detection and provide three additional advantages, namely, rapidly obtaining large amounts of RNA from samples, quickly detecting infective or pathogenic genes, and speedily confirming the detected exogenous genes. This application might be useful for detecting viral RNA and for the diagnosis of RNA virus-mediated diseases.

Hacking CD/DVD/Blu-ray for Biosensing.

The optical pickup unit (OPU) within a CD/DVD/Blu-ray drive integrates 780, 650, and 405 nm wavelength lasers, diffraction-limited optics, a high-bandwidth optoelectronic transducer up to 400 MHz, and a nanoresolution x-, z-axis, and tilt actuator in a compact size. In addition, the OPU is a remarkable piece of engineering and could enable different scientific applications such as sub-angstrom displacement sensing, micro- and nanoimaging, and nanolithography. Although off-the-shelf OPUs can be easily obtained, manufacturers protect their datasheets under nondisclosure agreements to impede their availability to the public. Thus, OPUs are black boxes that few people can use for research, and only experienced researchers can access all their functions. This review details the OPU mechanism and components. In addition, we explain how to utilize three commercially available triple-wavelength OPUs from scratch and optimize sensing quality. Then, we discuss scientific research using OPUs, from standard optical drive-based turnkey-biomarker array reading and OPU direct bioapplications (cytometry, optical tweezing, bioimaging) to modified OPU-based biosensing (DNA chip fluorescence scanning, biomolecular diagnostics). We conclude by presenting future trends on optical storage devices and potential applications. Hacking low-cost and high-performance OPUs may spread micro- and nanoscale biosensing research from research laboratories to citizen scientists around the globe.