Applications In Molecular Biology Research Articles

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii.

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

Comparative Analysis of 1,9-Dimethyl-Methylene Blue and Toluidine Blue Interactions with ct-DNA, G-Quadruplex DNA, and ssDNA

This study presents a comprehensive investigation into the interactions of two distinct synthetic dyes—1,9-Dimethyl-Methylene Blue and Toluidine Blue —with different DNA structures, namely calf thymus DNA (ct-DNA), G-quadruplex DNA, and single stranded DNA (ssDNA). The objective of this research is to elucidate the molecular affinities of these dyes for specific DNA structures and explore their potential applications in molecular biology and diagnostics. The experimental approach involved detailed UV-visible spectroscopic analyses, to probe the binding affinities of each dye with ct-DNA, G-quadruplex DNA, and ssDNA. The study aimed to assess the selectivity of these dyes towards the unique structural features of each DNA entity. The binding stoichiometry is defined from Job’s method. The selectivity of the dyes towards DNA also investigated with competitive dialysis experiments. The binding stoichiometry were 1:1 for 1,9-Dimethyl-Methylene Blue and Toluidine Blue and G-quadruplex DNA or ssDNA. Besides, results indicate that 1,9-Dimethyl-Methylene Blue and Toluidine Blue exhibit a pronounced affinity for G-quadruplex DNA, and ct-DNA. While single-stranded DNA is a fundamental component of DNA replication and transcription, our dyes exhibit lower affinity for this structure. The selectivity is advantageous, as it allows for the discrimination between single-stranded and structured DNA regions. The potential applications in studying DNA dynamics and unwinding processes are vast.

Research Progress on Liposome Pulmonary Delivery of Mycobacterium tuberculosis Nucleic Acid Vaccine and Its Mechanism of Action.

Traditional vaccines have played an important role in the prevention and treatment of infectious diseases, but they still have problems such as low immunogenicity, poor stability, and difficulty in inducing lasting immune responses. In recent years, the nucleic acid vaccine has emerged as a relatively cheap and safe new vaccine. Compared with traditional vaccines, nucleic acid vaccine has some unique advantages, such as easy production and storage, scalability, and consistency between batches. However, the direct administration of naked nucleic acid vaccine is not ideal, and safer and more effective vaccine delivery systems are needed. With the rapid development of nanocarrier technology, the combination of gene therapy and nanodelivery systems has broadened the therapeutic application of molecular biology and the medical application of biological nanomaterials. Nanoparticles can be used as potential drug-delivery vehicles for the treatment of hereditary and infectious diseases. In addition, due to the advantages of lung immunity, such as rapid onset of action, good efficacy, and reduced adverse reactions, pulmonary delivery of nucleic acid vaccine has become a hot spot in the field of research. In recent years, lipid nanocarriers have become safe, efficient, and ideal materials for vaccine delivery due to their unique physical and chemical properties, which can effectively reduce the toxic side effects of drugs and achieve the effect of slow release and controlled release, and there have been a large number of studies using lipid nanocarriers to efficiently deliver target components into the body. Based on the delivery of tuberculosis (TB) nucleic acid vaccine by lipid carrier, this article systematically reviews the advantages and mechanism of liposomes as a nucleic acid vaccine delivery carrier, so as to lay a solid foundation for the faster and more effective development of new anti-TB vaccine delivery systems in the future.

Role of Genome Sequences of Major and Minor Millets in Strengthening Food and Nutritional Security for Future Generations

Millets are small-seeded cereals belonging to the family Poaceae. They are considered to be climate-resilient and future nutritional food cereals for humans. Millets are resistant to biotic and abiotic stressors compared to other major cereals and thrive in low-quality soils with little maintenance and less rainfall. The importance of millets is still not well known to many people due to the lack of popularity and cultivation in semi-arid tropics of Asia and Africa. The United Nations has declared 2023 as the International Year of Millets (IYM 2023) to promote millet cultivation and popularize their health benefits globally. A few years ago, the application of molecular biology was in its infancy in millets due to the unavailability of genome sequences. Genome sequences are available for most of the millets on NCBI and Phytozome databases. In this review, we discuss the details of genome sequences for millets, candidate genes identified from the native genome of millets. The current status of quantitative trait loci and genome-wide association studies in millets are also discussed. The utilization of millet genome sequences in functional genomics research and translating the information for crop improvement will help millet and non-millet cereals survive harsh environments in the future. Such efforts will help strengthen food security and reduce malnutrition worldwide in 2050.

Purified Pyropia yezoensis Pigment Extract-Based Tandem Dye Synthesis.

Red phycoerythrin (R-PE) is a highly valuable protein found in an edible seaweed, Pyropia yezoensis. It is used extensively in biotechnological applications due to its strong fluorescence and stability in diverse environments. However, the current methods for extracting and purifying R-PE are costly and unsustainable. The aim of the present study was to enhance the financial viability of the process by improving the extraction and purification of R-PE from dried P. yezoensis and to further enhance R-PE value by incorporating it into a tandem dye for molecular biology applications. A combination of ultrafiltration, ion exchange chromatography, and gel filtration yielded concentrated (1 mg·mL-1) R-PE at 99% purity. Using purified PE and Cyanine5 (Cy5), an organic tandem dye, phycoerythrin-Cy5 (PE-Cy5), was subsequently established. In comparison to a commercially available tandem dye, PE-Cy5 exhibited 202.3% stronger fluorescence, rendering it suitable for imaging and analyzes that require high sensitivity, enhanced signal-to-noise ratio, broad dynamic range, or shorter exposure times to minimize potential damage to samples. The techno-economic analysis confirmed the financial feasibility of the innovative technique for the extraction and purification of R-PE and PE-Cy5 production.

Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR

Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10−5 bp−1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity – an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

Cold-active microbial enzymes and their biotechnological applications.

Microorganisms known as psychrophiles/psychrotrophs, which survive in cold climates, constitute majority of the biosphere on Earth. Their capability to produce cold-active enzymes along with other distinguishing characteristics allows them to survive in the cold environments. Due to the relative ease of large-scale production compared to enzymes from plants and animals, commercial uses of microbial enzyme are alluring. The ocean depths, polar, and alpine regions, which make up over 85% of the planet, are inhabited to cold ecosystems. Microbes living in these regions are important for their metabolic contribution to the ecosphere as well as for their enzymes, which may have potential industrial applications. Cold-adapted microorganisms are a possible source of cold-active enzymes that have high catalytic efficacy at low and moderate temperatures at which homologous mesophilic enzymes are not active. Cold-active enzymes can be used in a variety of biotechnological processes, including food processing, additives in the detergent and food industries, textile industry, waste-water treatment, biopulping, environmental bioremediation in cold climates, biotransformation, and molecular biology applications with great potential for energy savings. Genetically manipulated strains that are suitable for producing a particular cold-active enzyme would be crucial in a variety of industrial and biotechnological applications. The potential advantage of cold-adapted enzymes will probably lead to a greater annual market than for thermo-stable enzymes in the near future. This review includes latest updates on various microbial source of cold-active enzymes and their biotechnological applications.

Transcriptome Data Analysis Applied to Grapevine Growth Stage Identification

In agricultural production, it is fundamental to characterize the phenological stage of plants to ensure a good evaluation of the development, growth and health of crops. Phenological characterization allows for the early detection of nutritional deficiencies in plants that diminish the growth and productive yield and drastically affect the quality of their fruits. Currently, the phenological estimation of development in grapevine (Vitis vinifera) is carried out using four different schemes: Baillod and Baggiolini, Extended BBCH, Eichhorn and Lorenz, and Modified E-L. Phenological estimation requires the exhaustive evaluation of crops, which makes it intensive in terms of labor, personnel, and the time required for its application. In this work, we propose a new phenological classification based on transcriptional measures of certain genes to accurately estimate the stage of development of grapevine. There are several genomic information databases for Vitis vinifera, and the function of thousands of their genes has been widely characterized. The application of advanced molecular biology, including the massive parallel sequencing of RNA (RNA-seq), and the handling of large volumes of data provide state-of-the-art tools for the determination of phenological stages, on a global scale, of the molecular functions and processes of plants. With this aim, we applied a bioinformatic pipeline for the high-throughput quantification of RNA-seq datasets and further analysis of gene ontology terms. We identified differentially expressed genes in several datasets, and then, we associated them with the corresponding phenological stage of development. Differentially expressed genes were classified using count-based expression analysis and clustering and annotated using gene ontology data. This work contributes to the use of transcriptome data and gene expression analysis for the classification of development in plants, with a wide range of industrial applications in agriculture.

Speed meets accuracy: Advanced deep learning for efficient Orientia tsutsugamushi bacteria assessment in RNAi screening

This study investigates the use of advanced computer vision techniques for assessing the severity of Orientia tsutsugamushi bacterial infectivity. It uses fluorescent scrub typhus images obtained from molecular screening, and addresses challenges posed by a complex and extensive image dataset, with limited computational resources. Our methodology integrates three key strategies within a deep learning framework: transitioning from instance segmentation (IS) models to an object detection model; reducing the model's backbone size; and employing lower-precision floating-point calculations. These approaches were systematically evaluated to strike an optimal balance between model accuracy and inference speed, crucial for effective bacterial infectivity assessment. A significant outcome is that the implementation of the Faster R-CNN architecture, with a shallow backbone and reduced precision, notably improves accuracy and reduces inference time in cell counting and infectivity assessment. This innovative approach successfully addresses the limitations of image processing techniques and IS models, effectively bridging the gap between sophisticated computational methods and modern molecular biology applications. The findings underscore the potential of this integrated approach to enhance the accuracy and efficiency of bacterial infectivity evaluations in molecular research.

A Fusion of Taq DNA Polymerase with the CL7 Protein from Escherichia coli Remarkably Improves DNA Amplification.

DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a variant of CE7 (Colicin E7 DNase) from Escherichia coli. The resulting novel recombinant open reading frame was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 nM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.

Production of reverse transcriptase from Moloney murine Leukemia virus in Escherichia coli expression system

Reverse transcriptase (RT) is one of the most important enzymes used in molecular biology applications, enabling the conversion of RNA into complementary DNA (cDNA) that is used in reverse transcription-polymerase chain reaction (RT-PCR). The high demand of RT enzymes in biotechnological applications making the production optimization of RT is crucial for meeting the growing demand in industrial settings. Conventionally, the expression of recombinant RT is T7-induced promoter using IPTG in Escherichia coli expression systems, which is not cost-efficient. Here, we successfully made an alternative procedure for RT expression from Moloney murine leukemia virus (M-MLV) using autoinduction method in chemically defined medium. The optimization of carbon source composition (glucose, lactose, and glycerol) was analyzed using Response Surface Methodology (RSM). M-MLV RT was purified for further investigation on its activity. A total of 32.8 mg/L purified M-MLV RT was successfully obtained when glucose, glycerol, and lactose were present at concentration of 0.06%, 0.9%, and 0.5% respectively, making a 3.9-fold improvement in protein yield. In addition, the protein was produced in its active form by displaying 7462.50 U/mg of specific activity. This study provides the first step of small-scale procedures of M-MLV RT production that make it a cost-effective and industrially applicable strategy.

Development and validation of a 23-gene expression signature for molecular subtyping of medulloblastoma in a long-term Chinese cohort.

Medulloblastoma is the most common childhood malignant brain tumor andis a leading cause of cancer-related death in children. Recent transcriptional studies have shown that medulloblastomas comprise at least four molecular subgroups, each with distinct demographics, genetics, and clinical outcomes. Medulloblastoma subtyping has become critical for subgroup-specific therapies. The use of gene expression assays to determine the molecular subgroup of clinical specimens is a long-awaited application of molecular biology for this pediatric cancer. In the current study, we established a medulloblastoma transcriptome database of 460 samples retrieved from three published datasets (GSE21140, GSE37382, and GSE37418). With this database, we identified a 23-gene signature that is significantly associated with the medulloblastoma subgroups and achieved a classification accuracy of 95.2%. The 23-gene signature was further validated in a long-term cohort of 142 Chinese medulloblastoma patients. The 23-gene signature classified 21 patients as WNT (15%), 41 as SHH (29%), 16 as Group 3 (11%), and 64 as Group 4 (45%). For patients of WNT, SHH, Group 3, and Group 4, 5-year overall-survival rate reached 80%, 62%, 27%, and 47%, respectively (p < 0.0001), meanwhile 5-year progression-free survival reached 80%, 52%, 27%, and 45%, respectively (p < 0.0001). Besides, SHH/TP53-mutant tumors were associated with worse prognosis compared with SHH/TP53 wild-type tumors and other subgroups. We demonstrated that subgroup assignments by the 23-gene signature and Northcott's NanoString assay were highly comparable with a concordance rate of 96.4%. In conclusion, we present a novel gene signature that is capable of accurately and reliably assigning FFPE medulloblastoma samples to their molecular subgroup, which may serve as an auxiliary tool for medulloblastoma subtyping in the clinic. Future incorporation of this gene signature into prospective clinical trials is warranted to further evaluate its clinical.

Exploring the Impact of Primer-Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity.

Polymerase chain reaction (PCR) is a widely used technique in gene expression analysis, diagnostics, and various molecular biology applications. However, the accuracy and sensitivity of PCR can be compromised by primer-template mismatches, potentially leading to erroneous results. In this study, we strategically designed 111 primer-template combinations with varying numbers, types, and locations of mismatches to meticulously assess their impact on qPCR performance while two distinctly different types of DNA polymerases were used. Notably, when a single-nucleotide mismatch occurred at the 3' end of the primer, we observed significant decreases in the analytical sensitivity (0-4%) with Invitrogen™ Platinum™ Taq DNA Polymerase High Fidelity, while the analytical sensitivity remained unchanged with Takara Ex Taq Hot Start Version DNA Polymerase. Leveraging these findings, we designed a highly specific PCR to amplify Babesia while effectively avoiding the genetically close Theileria. Through elucidating the critical interplay between types of DNA polymerases and primer-template mismatches, this research provides valuable insights for improving PCR accuracy and performance. These findings have important implications for researchers aiming to achieve robust qPCR results in various molecular biology applications.

A comprehensive exploration of restriction enzymes and their applications in molecular biology: A review

Restriction enzymes, derived from bacteria, serve as indispensable tools in molecular biology, enabling precise manipulation of DNA with specificity. These enzymes recognize specific DNA sequences, cleaving the DNA strand at those designated sites. Two types, blunt-end cutters, and sticky-end producers, offer distinct advantages and disadvantages. In molecular biology, restriction enzymes find diverse applications, prominently in gene cloning, facilitating the insertion of foreign DNA into host organisms. Additionally, they play a vital role in DNA sequencing, DNA fingerprinting, and other techniques crucial for studying gene function and regulation. A groundbreaking development is witnessed in gene editing, where engineered enzymes recognize specific DNA sequences, allowing scientists to target and modify genes with unprecedented precision. This breakthrough holds the potential to revolutionize medicine, paving the way for treating genetic diseases and creating personalized therapies. Research efforts are focused on discovering new restriction enzymes with novel specificities, expanding the range of manipulable DNA sequences. This opens avenues for innovative applications in synthetic biology and biotechnology, further advancing the field. Despite their numerous applications, challenges persist, including the potential for off-target effects and the quest for enzymes with specific recognition sequences. Ongoing research and development continue to push the boundaries of what is achievable with restriction enzymes. In conclusion, restriction enzymes have significantly impacted molecular biology and biotechnology, facilitating the precise manipulation and study of genetic material. Ongoing research promises to unveil new applications and discoveries in this dynamic and promising field.

Fungal luminescence pathways: research and applications

The fungal bioluminescence pathway (FBP) is a metabolic pathway responsible for the generation of bioluminescence derived from fungi. This pathway utilizes caffeic acid as the substrate, generating a high-energy intermediate, and the decomposition of which yields green fluorescence with a wavelength of approximately 520 nm. The FBP is evolutionally conserved in luminescent fungal groups. Unlike other bioluminescent systems, the FBP is particularly suitable for engineering applications in eukaryotic organisms, especially in plants. Currently, metabolically engineered luminescent plants are able to emit visible light to illuminate its surroundings, which can be visualized clearly in the dark. The fungal bioluminescent system could be explored in various applications in molecular biology, biosensors and glowing ornamental plants, and even green lighting along city streets.

Multi-enzymatic systems synergize new RCA technique amplified super-long dsDNA from DNA circle

BackgroundIn the field of DNA amplification, there are great challenges in the effectively amplify of long-chain amplification, especially amplification up to several hundred kb level. ResultsA novel technique for the unbiased whole genome amplification from a thimbleful of DNA circles, such as low as 10 ng/ 10 μL of the circular cpDNA or low as 5 ng/ 10 μL of the plasmid, is developed, which can amplify an abundance of the whole genome sequences. Specifically, the new technique that combines rolling-amplification and triple-enzyme system presents a tightly controlled process of a series of buffers/reactions and optimized procedures, that applies from the primer-template duplexes to the Elution step. The result of this technique provides a new approach for extending RCA capacity, where it can reach 200 kb from the circular cpDNA amplification and 150 kb from the plasmid DNA amplification, that demonstrates superior breadth and evenness of genome coverage, high reproducibility, small amplification bias with the amplification efficiency. Significance and noveltyThis new technique will develop into one of the powerful tools for isothermal DNA amplification in vitro, genome sequencing/analysis, phylogenetic analysis, physical mapping, and other molecular biology applications.

Molecular Biology Applications of Psychrophilic Enzymes: Adaptations, Advantages, Expression, and Prospective.

Psychrophilic enzymes are primarily produced by microorganisms from extremely low-temperature environments which are known as psychrophiles. Their high efficiency at low temperatures and easy heat inactivation property have attracted extensive attention from various food and industrial bioprocesses. However, the application of these enzymes in molecular biology is still limited. In a previous review, the applications of psychrophilic enzymes in industries such as the detergent additives, the food additives, the bioremediation, and the pharmaceutical medicine, and cosmetics have been discussed. In this review, we discuss the main cold adaptation characteristics of psychrophiles and psychrophilic enzymes, as well as the relevant information on different psychrophilic enzymes in molecular biology. We summarize the mining and screening methods of psychrophilic enzymes. We finally recap the expression of psychrophilic enzymes. We aim to provide a reference process for the exploration and expression of new generation of psychrophilic enzymes.

Response strategies and biological applications of organic fluorescent thermometry: cell- and mitochondrion-level detection.

Temperature homeostasis is critical for cells to perform their physiological functions. Among the diverse methods for temperature detection, fluorescent temperature probes stand out as a proven and effective tool, especially for monitoring temperature in cells and suborganelles, with a specific emphasis on mitochondria. The utilization of these probes provides a new opportunity to enhance our understanding of the mechanisms and interconnections underlying various physiological activities related to temperature homeostasis. However, the complexity and variability of cells and suborganelles necessitate fluorescent temperature probes with high resolution and sensitivity. To meet the demanding requirements for intracellular/subcellular temperature detection, several strategies have been developed, offering a range of options to address this challenge. This review examines four fundamental temperature-response strategies employed by small molecule and polymer probes, including intramolecular rotation, polarity sensitivity, Förster resonance energy transfer, and structural changes. The primary emphasis was placed on elucidating molecular design and biological applications specific to each type of probe. Furthermore, this review provides an insightful discussion on factors that may affect fluorescent thermometry, providing valuable perspectives for future development in the field. Finally, the review concludes by presenting cutting-edge response strategies and research insights for mitigating biases in temperature sensing.

Precise Synthesis of Tetrablock Copolymers of Different Acrylamide Derivatives via Iterative Aqueous Cu(0)-Mediated Polymerization

The accessibility of specific synthesis of tetrablock copolymers is reported. In a specific synthesis, four acrylamide monomers are gradually added using an iterative aqueous Cu(0)-mediated reversible-deactivation radical polymerization (RDRP) method. Essential to the success of this approach is the ability to design and polymerize ABCD copolymer sequence with no need for immediate purification steps. The simple in-situ sequential polymerization method allowed for essentially perfect control of accurately well-defined tetrablock copolymers, which are composed of four tiny blocks, each of which contains an average of ten functional monomer units of acrylamide derivatives, resulting in a variety of functional groups. While the final molecular weight distributions have very narrow despersities (Đ &lt; 1.10), the efficient successive chain extension polymerization proceeded with high monomer conversions (&gt;99%), delivering excellent block purification in a short period of time. The tetrablock poly(NIPAM-DMA-HEAA-DEA) was characterized by NMR and GPC and showed beneficial end-group fidelity, allowing quantitative monitoring of the system’s alive nature after each synthetic cycle. Importantly, these one-pot syntheses are carried out at a below temperature of 0.0 °C in water as the solvent and can be implemented for applications of molecular biology. We also investigate the potential for a copper-amide complex to develop with acrylamide monomer, which could have an adverse effect on the end group’s functioning. Finally, we believe that this approach makes it easier to create a novel category of advanced polymeric materials.

Nucleobase-Modified Microgels Synthesized via Microfabrication Technology for DNA Adsorption

DNA isolation is a crucial procedure since DNA-based assays have great importance in molecular biology, biochemistry and biomedical applications. The objective of this study is to fabricate micron-sized hydrogels as adsorbents for DNA. Poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microgels were synthesized by free radical polymerization in the presence of N,N'-methylenebisacrylamide as a crosslinker, in the microholes of a microstencil array chip. Then, adenine was immobilized to microgels through the epoxy groups of glycidyl methacrylate. Scanning electron microscopy and Fourier transform infrared spectroscopy were employed to investigate the chemical and morphological characterizations of the microgels. The findings of the experiments demonstrate that the microgels had a cylindrical shape, were of uniform size, and had a height and diameter of around 500 μm. Observation of aromatic C=C peak confirmed the existence of adenine ligand in the microgel structure. Adsorption studies were carried out to determine the optimal conditions for DNA adsorption of nucleobase-immobilized microgels. After initially increasing, the quantity of DNA adsorbed onto the microgels reached a saturation level at a DNA concentration of around 2.0 mg/mL. The maximum adsorption was 38.54 mg/g microgels for an initial DNA concentration of 2.0 mg/mL in the optimum medium pH and temperature. DNA adsorption capabilities are shown to not significantly decline in recurrent adsorption-desorption cycles. As a result of the findings, adenine-immobilized microgels were demonstrated to be a viable option for DNA adsorption. Additionally, as a reference for future research, this study highlights the benefits of microfabrication technology, such as its simplicity of use in fabricating adsorption materials with the desired size, shape, and uniformity.