The BeadChip System: A Flexible Array Format for Complex Nucleic Acid and Protein Analysis

Nucleic acid analysis technology is the key to cracking the genetic information of life, which is very important for insight into disease diagnosis, drug development, food safety and environmental monitoring. The successful implementation of nucleic acid analysis depends on efficient and accurate nucleic acid sample purification technology. Traditional nucleic acid extraction methods are not only time-consuming and difficult to handle but also require skilled operators. Nanotechnology is gradually innovating nucleic acid extraction, simplifying the process and promoting biological science into a new era. The interaction modes between nanomaterials and nucleic acid molecules are diverse, including electrostatic interaction, covalent binding (direct covalent bonding, biotin-avidin system, disulfide bond connection, coordination bond, azide-alkyne click reaction, EDC/NHS coupling), π-π stacking effect, hydrogen bond formation, hydrophobic interaction and ion exchange. Among them, electrostatic interaction and covalent binding are particularly common and widely used. In addition, integrating nanomaterials into advanced monitoring systems such as microfluidic chips and biosensors provides strong support for the innovation of nucleic acid detection technology. The purpose of this paper is to comprehensively explain the basic principles and related molecular mechanisms of the interaction between nucleic acids and nanomaterials and to demonstrate their effectiveness in practical applications through specific examples for each interaction mode. Finally, we will review the latest progress of nanomaterial application in nucleic acid analysis, aiming to provide valuable references and inspirations for future research and development in this field.

Electrophoresis forms the basis of routine separation and analysis of nucleic acid and protein molecules. It is difficult to scan the biological literature without some form of electrophoresis being used in the methods and results for critical preparation and analysis. The applications of nucleic acid electrophoresis include purification, genetic analysis, DNA probe preparation, and DNA and RNA sequence determination. Protein electrophoresis is used for purification, size estimation, subunit composition, protein expression, and identification of posttranslational modifications, in addition to forming the basis for the first step in immunoblotting. While the analysis of proteins and nucleic acids is described in detail, the future applications of microelectrophoresis and microfluidics are also discussed. The bibliography contains both historically significant references and current references outlining established protocols, providing the bench user a guide to the use of electrophoretic techniques in their research.

Renewable microcolumns for solid-phase nucleic acid separations and analysis from environmental samples

Integrated slip valve-assisted fluidic chip coupling with CRISPR/Cas12a system for nucleic acid analysis

Nucleic acids are essential components of life. Both RNA and DNA are carriers of genetic information that are ultimate fundaments of life. Nucleic acids have specific structures and subcellular locations. Various laboratory techniques have been developed for the isolation of nucleic acids and the analysis of quality and quantity of the final product. A similar plethora of procedures has been developed to determine the base sequence of nucleic acids. These methods have been transferred into molecular assays that can be used for diagnostic purposes. The most important techniques are hybridisation, DNA amplification and DNA sequence analysis.

Future Analytical Techniques: DNA Mass Spectrometry

Nucleic acid analysis is used in many areas of life sciences such as medicine, food safety, and environmental monitoring. Accurate, reliable measurements of nucleic acids are crucial for maximum impact, yet users are often unaware of the global metrological infrastructure that exists to support these measurements. In this work, we describe international efforts to improve nucleic acid analysis, with a focus on the Nucleic Acid Analysis Working Group (NAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM). The NAWG is an international group dedicated to improving the global comparability of nucleic acid measurements; its primary focus is to support the development and maintenance of measurement capabilities and the dissemination of measurement services from its members: the National Metrology Institutes (NMIs) and Designated Institutes (DIs). These NMIs and DIs provide DNA and RNA measurement services developed in response to the needs of their stakeholders. The NAWG members have conducted cutting edge work over the last 20 years, demonstrating the ability to support the reliability, comparability, and traceability of nucleic acid measurement results in a variety of sectors.

Central to genomic research is the analysis of nucleic acids, which requires versatile, sensitive and high throughput technologies that can deliver accurate results in a short time. Reported herein are recent developments for sizing and/or quantifying nucleic acids (dsDNA and RNA) on a parallel capillary electrophoresis platform with LED-based fluorescence detection (AdvanCE™ FS system). This flexible multi-capillary platform has been employed for applications such as SSR, gDNA analysis, RNA analysis, quantification of next generation sequencing (NGS) libraries, and the separation and sizing of large cDNA from Populus spp,This poster will discuss recent validation of the platform for a subset of these applications in detail. The AdvanCE™ FS system successfully scored all SSR samples with resolution as low as 2 bp. Assessment of the quality/concentration of gDNA is also demonstrated with a new separation matrix, for separation of large DNA fragments. Separation and sizing of large cDNA was also demonstrated with good resolution and accuracy. A method for rapid and sensitive detection of total RNA concentration and quality will be discussed, as well as the sizing and quantification of NGS libraries.The AdvanCE™ FS platform offers rapid separation and ample resolution with excellent sensitivity and dynamic range, to benefit a variety of applications in genomic research.

Electrophoresis was introduced by Tiselius in 1937. He received the Nobel Prize for his work that demonstrated that protein solutions migrate in a direction and at a rate determined by their charge and mobility when an electrical current is applied. Slab gel electrophoresis (SGE) is an adaptation of this technique and has been used for decades as the primary tool for purification and analysis of nucleic acids and proteins. Gels are typically made of cross—linked polyacrylamide or agarose, and the separations are carried out in low electric fields. While many slab gel techniques are well established and effective in the analysis of nucleic acids, accurate quantitation and ease of automation remain obstacles.

On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

An internal amplification control for quantitative nucleic acid analysis using nanoparticle-based dipstickbiosensors

Chemistry at interfaces and in transport.- Lipase structures at the interface between chemistry and biochemistry.- Structure and orientation of hydrophobic surfactant-associated proteins in a lipid environment.- Enzyme function in organic solvents.- Protein sorting signals: Simple peptides with complex functions.- Chemistry and biochemistry.- Enzymes: Chemistry and biochemistry.- Analysis of the structure of naturally processed peptides bound by Class I and Class II major histocompatibility complex molecules.- Catalytic antibodies: Evolution of protein function in real time.- Analysis of proteins and nucleic acids.- Chemical techniques employed for the primary structural analysis of proteins and peptides.- UV and nucleic acids.- Synthesis of active compounds.- Chemical and enzymatic synthesis of glycopeptides.- Peptides as active probes.- Metalloproteins.- Zinc metallochemistry in biochemistry.- NMR Structural studies on the zinc finger domains of nuclear hormone receptors.

Method verification or validation studies using quantitative standards are required for diagnostic tests to be used in microbiological diagnostic laboratories. Standards containing whole viruses instead of synthetic nucleic acids or plasmids ensures that all steps of the assay, including extraction, reverse transcription and polymerase chain reaction (PCR), are evaluated as in real life. Commercial quantitative standard materials for nucleic acid tests are limited for respiratory viruses. In this study, we aimed to develop quantitative nucleic acid standards for influenza A (infA), influenza B (infB) viruses and respiratory syncytial virus (RSV) using digital PCR (dPCR). In the study, the amount of viral nucleic acids in samples prepared by pooling nasopharyngeal swab samples known to be positive for RSV, infA, infB RNA were determined by dPCR (QIAcuity, Qiagen) using commercial primer/probe sets (Qiagen, Germany). Nucleic acid extraction was performed using a commercial kit (Xi’an Tianlong Science&Technology Co, China). The analytical sensitivity (LoD) and lower limit of quantitation (LoQ), intra- and inter-study reproducibility and linearity of the dPCR method for infA, infB and RSV were determined. Samples were analysed by both dPCR and real-time RT-PCR (qRT-PCR) and the relationship between Ct values and dPCR quantification results was evaluated by linear regression. Statistical analysis were performed using GraphPad Prism 10.4.0 (GraphPad, USA) and Excel Analysis ToolPak. The LoD values of the dPCR method for infA, infB and RSV were 93.75, 15.59 and 26.23 copies/mL, respectively. The intra-study reproducibility (coefficient of variation, CV%) of the dPCR method ranged from 0.06 to 7.97, being higher in samples with low viral load. Inter-study reproducibility was 0.73 to 5.41. Linearity analysis performed with dilutions in the range of 3-4 log10 for infA and in infB and seven log10 for RSV showed r2≥ 0.99 for all three viruses. The concentrations measured by dPCR were correlated with qRT-PCR Ct results. When the intra- and inter-study reproducibility results of dPCR and qRT-PCR tests were compared, the CV% value of dPCR was significantly lower (p= 0.0312). The results of the study showed that the dPCR method is a highly reproducible and reliable method for obtaining quantitative standards. The quantitative standards obtained can be used to develop assays for viral load determination and/or to perform method confirmation analysis of such assays. In conclusion, in this study reliable quantitative nucleic acid standards for infA, infB and RSV were obtained by dPCR using pooled patient samples and performance analysis of the dPCR method was determined. This study was an example of the production of quantitative viral nucleic acid standards by dPCR.

The numerous problems posed by modern biochemistry, biology, and medicine, as well as the growing significance of genetic engineering require the application of fast and reliable methods of utmost sensitivity and selectivity for the analysis of nucleic acids. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) represent established analytical techniques for the characterization and structural elucidation of single- and double-stranded nucleic acids, ranging in size from a few nucleotides to several thousand base pairs. Although both techniques are independently applicable for nucleic acid analysis, the on-line hyphenation significantly enhances their potential for the robust and fully automable routine analysis of minute amounts of biological samples. Among the various chromatographic and mass spectrometric modes available in principle, ion-pair reversed-phase HPLC and electrospray ionization mass spectrometry (ESI-MS) have been shown to be the most suitable for the direct interfacing of liquid chromatography (LC) and MS. Instrumental setup, as well as chromatographic and mass spectrometric experimental conditions, need to be carefully selected in order to maximize the performance of the hyphenated analytical system. Applications of HPLC-ESI-MS include the characterization of oligodeoxynucleotides synthesized by solid-phase synthesis, the analysis of antisense oligodeoxynucleotides, oligonucleotide metabolites, and DNA adducts, the analysis of genomic segments specifically amplified by the polymerase chain reaction (PCR), the characterization of ribonucleic acids, the sizing of double-stranded DNA restriction fragments, the genotyping of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), the detection of mutations in nucleic acid sequences, and the sequencing of nucleic acids.

Detection and analysis of nucleic acid in various biological samples of COVID-19 patients