Rapid DNA Research Articles

Validation of the isothermal Schistosoma haematobium Recombinase Polymerase Amplification (RPA) assay, coupled with simplified sample preparation, for diagnosing female genital schistosomiasis using cervicovaginal lavage and vaginal self-swab samples.

BackgroundFemale genital schistosomiasis (FGS) is a neglected and disabling gynecological disease that can result from infection with the parasitic trematode Schistosoma haematobium. Accurate diagnosis of FGS is crucial for effective case management, surveillance and control. However, current methods for diagnosis and morbidity assessment can be inaccessible to those at need, labour intensive, costly and unreliable. Molecular techniques such as PCR can be used to reliably diagnose FGS via the detection of Schistosoma DNA using cervicovaginal lavage (CVL) samples as well as lesser-invasive vaginal self-swab (VSS) and cervical self-swab samples. PCR is, however, currently unsuited for use in most endemic settings. As such, in this study, we assessed the use of a rapid and portable S. haematobium recombinase polymerase amplification (Sh-RPA) isothermal molecular diagnostic assay, coupled with simplified sample preparation methodologies, to detect S. haematobium DNA using CVL and VSS samples provided by patients in Zambia.Methodology/Principal findingsVSS and CVL samples were screened for FGS using a previously developed Sh-RPA assay. DNA was isolated from VSS and CVL samples using the QIAamp Mini kit (n = 603 and 527, respectively). DNA was also isolated from CVL samples using two rapid and portable DNA extraction methods: 1) the SpeedXtract Nucleic Acid Kit (n = 223) and 2) the Extracta DNA Tissue Prep Kit (n = 136). Diagnostic performance of the Sh-RPA using VSS DNA extacts (QIAamp Mini kit) as well as CVL DNA extracts (QIAamp Mini kit, SpeedXtract Nucleic Acid Kit and Extracta DNA Tissue Prep Kit) was then compared to a real-time PCR reference test.Results suggest that optimal performance may be achieved when the Sh-RPA is used with PuVSS samples (sensitivity 93.3%; specificity 96.6%), however no comparisons between different DNA extraction methods using VSS samples could be carried out within this study. When using CVL samples, sensitivity of the Sh-RPA ranged between 71.4 and 85.7 across all three DNA extraction methods when compared to real-time PCR using CVL samples prepared using the QIAamp Mini kit. Interestingly, of these three DNA extraction methods, the rapid and portable SpeedXtract method had the greatest sensitivity and specificity (85.7% and 98.1%, respectively). Specificity of the Sh-RPA was >91% across all comparisons.Conclusions/SignificanceThese results supplement previous findings, highlighting that the use of genital self-swab sampling for diagnosing FGS should be explored further whilst also demonstrating that rapid and portable DNA isolation methods can be used to detect S. haematobium DNA within clinical samples using RPA. Although further development and assessment is needed, it was concluded that the Sh-RPA, coupled with simplified sample preparation, shows excellent promise as a rapid and sensitive diagnostic tool capable of diagnosing FGS at the point-of-care in resource-poor schistosomiasis-endemic settings.

Active DNA demethylation regulates MAMP-triggered immune priming in Arabidopsis

Plants recognize microbe-associated molecular patterns (MAMPs) to activate immune responses and defense priming to defend against pathogen infections. Transcriptional regulation of gene expression is crucial for plant immunity and is mediated by multiple factors, including DNA methylation. However, it remains unknown whether and how DNA demethylation contributes to immune responses in MAMP-triggered immunity. Here, we report that active DNA demethylation is required for MAMP-triggered immunity to bacterial pathogens. The rdd-2 triple mutant carrying mutations in ROS1, DML2, and DML3 that encode DNA glycosylases, which are key DNA demethylation enzymes, exhibits compromised immune responses triggered by the MAMPs flg22 and elf18. Genome-wide methylome analysis reveals that flg22 induces rapid and specific DNA demethylation in an RDD-dependent manner. The expression levels of salicylic acid signaling-related and phytoalexin biosynthesis-related genes are tightly associated with the flg22-induced promoter demethylation. The compromised accumulation of priming compounds and antimicrobial metabolites ultimately leads to a defense priming defect in the rdd-2 mutant. Our results reveal the critical role of active DNA demethylation in the MAMP-triggered immune response and provide unique insight into the molecular mechanism of flg22-modulated DNA demethylation.

Metal nanoclusters combined with CRISPR-Cas12a for hepatitis B virus DNA detection

Since hepatitis B virus (HBV) infection can cause chronic viral hepatitis, increase the risk of liver fibrosis and cirrhosis, and ultimately result in hepatoma, it is of great significance to explore convenient, rapid, and sensitive diagnostic strategies for HBV DNA detection. In this study, we developed a novel and sensitive fluorescent biosensor for HBV detection based on the sensing system consisting of CRISPR-Cas12a enzymes and metal nanoclusters as the luminescent nanoprobes, involving gold nanoclusters (AuNCs), silver nanoclusters (AgNCs), and copper nanoclusters (CuNCs). The DNA probes could effectively be applied as the template for the preparation of fluorescent metal nanoclusters. In the presence of the HBV target, the trans-cleavage capability of Cas12a was able to be initiated to degrade DNA probes, which brought about the inhibition of metal nanoclusters formation, thereby giving minimal fluorescence signals. In comparison with AuNCs and AgNCs, the CuNCs-based nanosensor exhibited much higher sensitivity. In addition, the fluorescence response of CuNCs to HBV detection was significantly faster, which could be completed within 25 min without the requirements of sophisticated equipment or harsh reaction conditions. Moreover, the practical application of this analysis method was also demonstrated to detect HBV DNA target in human serum samples with satisfactory recovery, which was promising for simple, rapid, sensitive, and label-free HBV DNA bioanalysis.

Rapid DNA Sequencing Technology Based on the Sanger Method for Bacterial Identification.

Antimicrobial resistance, a global health concern, has been increasing due to inappropriate use of antibacterial agents. To facilitate early treatment of sepsis, rapid bacterial identification is imperative to determine appropriate antibacterial agent for better therapeutic outcomes. In this study, we developed a rapid PCR method, rapid cycle sequencing, and microchip electrophoresis, which are the three elemental technologies for DNA sequencing based on the Sanger sequencing method, for bacterial identification. We achieved PCR amplification within 13 min and cycle sequencing within 14 min using a rapid thermal cycle system applying microfluidic technology. Furthermore, DNA analysis was completed in 14 min by constructing an algorithm for analyzing and performing microchip electrophoresis. Thus, the three elemental Sanger-based DNA sequencing steps were accomplished within 41 min. Development of a rapid purification process subsequent to PCR and cycle sequence using a microchip would help realize the identification of causative bacterial agents within one hour, and facilitate early treatment of sepsis.

A Cutoff Determination of Real-Time Loop-Mediated Isothermal Amplification (LAMP) for End-Point Detection of Campylobacter jejuni in Chicken Meat.

Campylobacter jejuni is one of the leading causes of foodborne illness worldwide. C. jejuni is commonly found in poultry. It is the most frequent cause of contamination and thus resulting in not only public health concerns but also economic impacts. To test for this bacterial contamination in food processing plants, this study attempted to employ a simple and rapid detection assay called loop-mediated isothermal amplification (LAMP). The best cutoff value for the positive determination of C. jejuni calculated using real-time LAMP quantification cycle (Cq) was derived from the receiver operating characteristic (ROC) curve modeling. The model showed an area under curve (AUC) of 0.936 (95% Wald CI: 0.903–0.970). Based on Youden’s J statistic, the optimal cutoff value which had the highest sensitivity and specificity from the model was calculated as 18.07. The LAMP assay had 96.9% sensitivity, 95.8% specificity, and 93.9 and 97.9% positive and negative predictive values, respectively, compared to a standard culture approach for C. jejuni identification. Among all non-C. jejuni strains, the LAMP assay gave each of 12.5% false-positive results to C. coli and E. coli (1 out of 8 samples). The assay can detect C. jejuni at the lowest concentration of 103 CFU/mL. Our results suggest a preliminary indicator for the application of end-point LAMP assays, such as turbidity and UV fluorescence tests, to detect C. jejuni in field operations. The LAMP assay is an alternative screening test for C. jejuni contamination in food samples. The method provides a rapid detection, which requires only 9 min with a cutoff value of Cq. We performed the extraction of DNA from pure cultures and the detection of C. jejuni using the LAMP assay within 3 h. However, we were not able to reduce the time for the process of enrichment involved in our study. Therefore, we suggest that alternative enrichment media and rapid DNA extraction methods should be considered for further study. Compared to other traditional methods, our proposed assay requires less equipment and time, which is applicable at any processing steps in the food production chain.

Structural basis for mismatch surveillance by CRISPR\u2013Cas9

CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1–4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5–7. Although Cas9 variants with greater discrimination against mismatches have been designed8–10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.

Rapid Diagnostic Test for Hepatitis B Virus Viral Load Based on Recombinase Polymerase Amplification Combined with a Lateral Flow Read-Out.

Hepatitis B (HBV) infection is a major public health concern. Perinatal transmission of HBV from mother to child represents the main mode of transmission. Despite the existence of effective immunoprophylaxis, the preventive strategy is inefficient in neonates born to mothers with HBV viral loads above 2 × 105 IU/mL. To prevent mother-to-child transmission, it is important to identify highly viremic pregnant women and initiate antiviral therapy to decrease their viral load. We developed a simple innovative molecular approach avoiding the use of automatic devices to screen highly viremic pregnant women. This method includes rapid DNA extraction coupled with an isothermal recombinase polymerase amplification (RPA) combined with direct visual detection on a lateral flow assay (LFA). We applied our RPA-LFA approach to HBV DNA-positive plasma samples with various loads and genotypes. We designed a triage test by adapting the analytical sensitivity to the recommended therapeutic decision threshold of 2 × 105 IU/mL. The sensitivity and specificity were 98.6% (95% CI: 92.7–99.9%) and 88.2% (95% CI: 73.4–95.3%), respectively. This assay performed excellently, with an area under the ROC curve value of 0.99 (95% CI: 0.99–1.00, p < 0.001). This simple method will open new perspectives in the development of point-of-care testing to prevent HBV perinatal transmission.

Targeting of RecQ Helicases as a Novel Therapeutic Strategy for Ovarian Cancer.

Simple SummaryOvarian cancer is the most lethal gynecologic malignancy and is characterized by genomic instability and DNA repair defects. PARP inhibitors (PARPi) changed the treatment paradigm of ovarian cancer but the development of resistance to PARPi is a pressing clinical challenge. In this review, we discuss how RecQ helicases can be targeted as a novel therapeutic strategy to prevent such treatment resistance. The combination of helicase inhibitor with a PARP inhibitor (PARPi) or ATR inhibitor may overcome PARPi resistance in ovarian cancer.RecQ helicases are essential for DNA replication, recombination, DNA damage repair, and other nucleic acid metabolic pathways required for normal cell growth, survival, and genome stability. More recently, RecQ helicases have been shown to be important for replication fork stabilization, one of the major mechanisms of PARP inhibitor resistance. Cancer cells often have upregulated helicases and depend on these enzymes to repair rapid growth-promoted DNA lesions. Several studies are now evaluating the use of RecQ helicases as potential biomarkers of breast and gynecologic cancers. Furthermore, RecQ helicases have attracted interest as possible targets for cancer treatment. In this review, we discuss the characteristics of RecQ helicases and their interacting partners that may be utilized for effective treatment strategies (as cancers depend on helicases for survival). We also discuss how targeting helicase in combination with DNA repair inhibitors (i.e., PARP and ATR inhibitors) can be used as novel approaches for cancer treatment to increase sensitivity to current treatment to prevent rise of treatment resistance.

Loop-Mediated Isothermal Amplification Coupled With Nanoparticle-Based Lateral Biosensor for Rapid, Sensitive, and Specific Detection of Bordetella pertussis.

Bordetella pertussis is the most frequent causative agent for pertussis, which is a highly contagious disease. Here, we developed a method based on loop-mediated isothermal amplification (LAMP) and nanoparticle-based lateral flow biosensor (LFB) for the timely diagnosis of B. pertussis infections. A set of six primers was designed for LAMP reactions, and the LAMP results were rapidly and visually indicated using LFB. The recommended condition for the B. pertussis LAMP reactions is 40 min at 66°C. Our results confirmed that the LAMP-LFB assay could specifically detect B. pertussis and did not cross-react with non-B. pertussis isolates. The sensitivity of the B. pertussis LAMP-LFB assay was 50 fg per reaction. In particular, 108 nasopharyngeal swab (NPS) samples were collected to evaluate the B. pertussis LAMP-LFB assay, and the results were compared with those of the quantitative PCR (qPCR) method. The positive rates of B. pertussis LAMP-LFB and qPCR were 40.7% and 38.8%, respectively, and the agreement between the LAMP-LFB and qPCR results was 98%, with a kappa value of 0.96. The whole process of LAMP-LFB can be completed within 1 h, which is much shorter than that of qPCR, including about 15 min of rapid DNA extraction, 40 min of LAMP reaction, and within 2 min of the LFB test. Collectively, the B. pertussis LAMP-LFB assay developed in this report offers a new option for the rapid, reliable, and simple diagnosis of B. pertussis infections.

Early differential responses elicited by BRAFV600E in adult mouse models

The BRAF gene is frequently mutated in cancer. The most common genetic mutation is a single nucleotide transition which gives rise to a constitutively active BRAF kinase (BRAFV600E) which in turn sustains continuous cell proliferation. The study of BRAFV600E murine models has been mainly focused on the role of BRAFV600E in tumor development but little is known on the early molecular impact of BRAFV600E expression in vivo. Here, we study the immediate effects of acute ubiquitous BRAFV600E activation in vivo. We find that BRAFV600E elicits a rapid DNA damage response in the liver, spleen, lungs but not in thyroids. This DNA damage response does not occur at telomeres and is accompanied by activation of the senescence marker p21CIP1 only in lungs but not in liver or spleen. Moreover, in lungs, BRAFV600E provokes an acute inflammatory state with a tissue-specific recruitment of neutrophils in the alveolar parenchyma and macrophages in bronchi/bronchioles, as well as bronchial/bronchiolar epithelium transdifferentiation and development of adenomas. Furthermore, whereas in non-tumor alveolar type II (ATIIs) pneumocytes, acute BRAFV600E induction elicits rapid p53-independent p21CIP1 activation, adenoma ATIIs express p53 without resulting in p21CIP1 gene activation. Conversely, albeit in Club cells BRAFV600E-mediated proliferative cue is more exacerbated compared to that occurring in ATIIs, such oncogenic stimulus culminates with p21CIP1-mediated cell cycle arrest and apoptosis. Our findings indicate that acute BRAFV600E expression drives an immediate induction of DNA damage response in vivo. More importantly, it also results in rapid differential responses of cell cycle and senescence-associated proteins in lung epithelia, thus revealing the early molecular changes emerging in BRAFV600E-challenged cells during tumorigenesis in vivo.

Scale-up issues for commercial depth filters in bioprocessing.

Significant increases in cell density and product titer have led to renewed interest in the application of depth filtration for initial clarification of cell culture fluid in antibody production. The performance of these depth filters will depend on the local pressure and velocity distribution within the filter capsule, but these are very difficult to probe experimentally, leading to challenges in both process design and scale-up. We have used a combination of carefully designed experimental studies and computational fluid dynamics (CFD) to examine these issues in both lab-scale (SupracapTM 50) and pilot-scale (StaxTM ) depth filter modules, both employing dual-layer lenticular PDH4 media containing diatomaceous earth. The SupracapTM 50 showed a more rapid increase in transmembrane pressure and a more rapid DNA breakthrough during filtration of a Chinese Hamster Ovary cell culture fluid. These results were explained using CFD calculations which showed very different flow distributions within the modules. CFD predictions were further validated using measurements of the residence time distribution and dye binding in both the lab-scale and pilot-plant modules. These results provide important insights into the factors controlling the performance and scale-up of these commercially important depth filters as well as a framework that can be broadly applied to develop more effective depth filters and depth filtration processes.

Recombinase polymerase amplification combined with fast DNA extraction for on–spot identification of Deinagkistrodon acutus, a threatened species

This study addresses the use of recombinase polymerase amplification combined with fast DNA extraction for on–spot identification of Deinagkistrodon acutus, a snake species threatened due to over–exploitation and habitat destruction. For its conservation, an efficient species identification method is urgently neededto fight against illegal capture and trade. Fourteen individuals representing 12 snake species (including D. acutus and other snake species) were collected from mountainous regions in Southern China. Genomic DNA was extracted within five minutes by a modified alkaline lysis method. Species–specific primers for recombinase polymerase amplification (RPA) were designed based on the sequences of cytochrome C oxidase subunit I (COI) barcode region, and an optimized RPA assay system was set up. Specificity and sensitivity of the assay were checked, and the assay was validated by identifying 10 commercial Qi She crude drug samples derived from D. acutus. Under optimized RPA conditions, a distinct single band of 354 bp was amplified only for D. acutus but not for the related snake species. The entire procedure can be completed in 30 min at room temperature. Commercial Qi She crude drug identification validated effectiveness of the established assay system. Using a recombinase polymerase amplification (RPA) assay with rapid DNA extraction, we established an on–spot D. acutus identification method with good specificity and sensitivity. This method could become an efficient tool for rigorous supervision of illegal D. acutus capture and trade.

Rapid Identification of DNA Fragments through Direct Sequencing with Electro-Optical Zero-Mode Waveguides.

In contrast to sequence-specific techniques such as polymerase chain reaction, DNA sequencing does not require prior knowledge of the sample for surveying DNA. However, current sequencing technologies demand high inputs for a suitable library preparation, which typically necessitates DNA amplification, even for single-molecule sequencing methods. Here, electro-optical zero-mode waveguides (eZMWs) are presented, which can load DNA into the confinement of zero-mode waveguides with high efficiency and negligible DNA fragment length bias. Using eZMWs, highly efficient voltage-induced loading of DNA fragments of various sizes from ultralow inputs (nanogram-to-picogram levels) is observed. Rapid DNA fragment identification is demonstrated by burst sequencing of short and long DNA molecules (260 and 20000bp) loaded from an equimolar picomolar-level concentration mixture in just a few minutes. The device allows further studies in which low-input DNA capture is essential, for example, in epigenetics, where native DNA is required for obtaining modified base information.

Analysis of mixed DNA profiles from the RapidHIT™ ID platform using probabilistic genotyping software STRmix™

Rapid DNA instruments are gaining interest in the forensic community as a means to generate DNA profile information more quickly than standard laboratory workflows, and with the potential to be carried out at the scene where samples are taken. Due to the many years that DNA profiles have been generated in a standard laboratory workflow, there have been numerous studies into profiling performance. These have flowed into probabilistic means of evaluating DNA profiles produced in those laboratories. In this study we examined the DNA profiling performance of the RapidHIT™ ID system on artificial mixtures constructed from raw DNA, mixtures constructed from body fluids, and touch DNA samples. We calibrated the probabilistic genotyping system STRmix™ for use on results produced by the RapidHIT ID system. Identical DNA samples were split, with half analysed on the RapidHIT ID system and the other half analysed in the standard laboratory workflow. Profiles produced from these paired samples were compared with regards to their composition and discrimination power. In general, profiles produced using the RapidHIT ID system showed good discrimination power, but less than those produced via the standard laboratory workflow. This is the expected trade-off for the advantages of speed and portability.

HMCES safeguards genome integrity and long-term self-renewal of hematopoietic stem cells during stress responses.

Hematopoietic stress drives quiescent hematopoietic stem cells (HSCs) to proliferate, generating reactive oxygen species (ROS) and oxidative DNA damage including abasic sites. Such a coupling between rapid DNA replication and a burst of abasic site formation during HSC stress responses, however, presents a challenge to accurately repair abasic sites located in replication-associated single-stranded DNA. Here we show that HMCES, a novel shield of abasic sites, plays pivotal roles in overcoming this challenge upon HSC activation. While HMCES was dispensable for steady-state hematopoiesis, Hmces-deficient HSCs exhibited compromised long-term self-renewal capacity in response to hematopoietic stress such as myeloablation and transplantation. Loss of HMCES resulted in accumulation of DNA lesions due to impaired resolution of abasic sites generated by activation-induced ROS in activated HSCs and broad downregulation of DNA damage response and repair pathways. Moreover, Hmces-deficient mice died from bone marrow failure after exposure to sublethal irradiation, which also produces ROS. Notably, dysregulation of HMCES occurs frequently in acute lymphocytic leukemia (ALL) and is associated with poor clinical outcomes. Together, our findings not only highlighted HMCES as a novel genome protector in activated HSCs, but also position it as a potential selective target against ALL while sparing normal hematopoiesis.

Analysis of rapid HIT application to touch DNA samples.

Rapid DNA technology is being utilized for reference profiles worldwide. There is also strong data in the literature to support its use for high-template DNA sources, the same is not true for low-template sources, such as touch DNA; this is a requirement before wider implementation to forensic casework is considered. We report on the Rapid HIT Intel cartridge's ability to facilitate successful amplification of touch DNA to obtain profiles from template deposited on items commonly encountered in forensic casework. Eight items were touched in ten replicates- two were tapelifted, three swabbed, and three directly inserted. Significance was observed in the alleles amplified and RFU with respect to sample type. Three samples performed well: cable tie, fabric, and matchstick. As two of these were directly inserted, this should be considered for any sample small enough. Placement of highly absorbent substrates into the cartridge is not advised as it can cause a lysate-pull error. Heterozygote loci often presented as homozygous (32%-78% loci per profile); this was influenced by substrate type and profile RFU. Loci with larger masses exhibited higher false homozygosity also. Comparison of the donor's profile analyzed was performed against previous datasets analyzing touch DNA through standard workflow, including manual DNA extraction, PCR, and CE separation. These data show that for all substrates, except for a fabric swatch, standard processing is preferential to Rapid HIT analysis. In its current form, rapid DNA technology is not fit for the routine analysis of touch DNA samples in forensic casework.

Identification of DNA nucleotides by conductance and tunnelling current variation through borophene nanogaps.

Rapid and inexpensive DNA sequencing is critical to biomedical research and healthcare for the accomplishment of personalized medicine. Solid-state nanopores and nanogaps have marshalled themselves in the fascinating paradigm of nano-research since the advent of its application in DNA sequencing by analyzing the quantum conductance and electric current signals. In this study, the feasibility of the considered borophene nanogaps for DNA sequencing purposes via the electronic tunnelling current approach was investigated by utilizing combined density functional theory with non-equilibrium Green's function (DFT-NEGF) techniques. The interaction energy (Ei) and the charge density difference (CDD) plots exploit the charge modulation around the nanogap edges due to the presence of each nucleotide. Our results revealed a distinct variation in the tunnelling conductance, as a characteristic fingerprint of each nucleotide at the Fermi level. The calculated tunnelling current variation across the nanogap under an applied bias voltage was also significant due to the effective coupling of nucleotides with the electrode edges. The current was in the picoampere (pA) range, which was fairly higher than the electrical background noise and also experimentally detectable by the canning tunnelling microscopy (STM) technique. Our findings demonstrated that in the borophene nanopore vs. nanogap scenario, the nanogap has several advantages and is a more promising nanobiosensor. Moreover, we also compared our results with various previous experimental and theoretical reports on nanogaps as well as nanopores for gaining better insights.

Establishing and Operating a Rapid DNA Reachback Center

Establishing and Operating a Rapid DNA Reachback Center

Bubble-enhanced ultrasonic microfluidic chip for rapid DNA fragmentation.

DNA fragmentation is an essential process in developing genetic sequencing strategies, genetic research, as well as for the diagnosis of diseases with a genetic signature like cancer. Efficient on-chip DNA fragmentation protocols would be beneficial to process integration and open new opportunities for microfluidics in genetic applications. Here we present an acoustic microfluidic chip comprising an array of ultrasound-actuated microbubbles located at dedicated positions adjacent to a channel containing the DNA sample solution. The efficiency of the on-chip DNA fragmentation process arises mainly from tensile forces generated by acoustic streaming near the oscillating bubble interfaces, as well as a synergistic effect of streaming stress and ultrasonic cavitation. Acoustic microstreaming and the pressure distribution in the DNA channel were assessed by finite element simulation. We characterized the bubble-enhanced effect by measuring gene fragment size distributions with respect to different ultrasound parameters. For optimized on-chip conditions, purified lambda (λ) DNA (48.5 kbp) could be disrupted to fragments with an average size of 2 kbp after 30 s and down to 300 bp after 90 s. Mouse genomic DNA (1.4 kbp) fragmentation size decreased to 500 bp in 30 s and reduced further to 250 bp in 90 s. Bubble-induced fragmentation was more than 3 times faster than without bubbles. On-chip performance and process yield were found to be comparable to a sophisticated high-end commercial system. In this view, our new bubble-enhanced microfluidic approach is a promising tool for current and next generation sequencing platforms with high efficiency and good capacity. Moreover, the availability of an efficient on-chip DNA fragmentation process opens perspectives for implementing full molecular protocols on a single microfluidic platform.

Illusion of Transcriptional Regulations by Rapid DNA Binding of a Regulator and Essential Requirement of Abortive Initiation for Regulations

Illusion of Transcriptional Regulations by Rapid DNA Binding of a Regulator and Essential Requirement of Abortive Initiation for Regulations