Magnetic Bead-based Extraction Research Articles

Advances in DNA Extraction Techniques: A Comprehensive Review of Methods and Applications

The extraction of DNA is a fundamental process in molecular biology, underpinning a wide range of applications from genetic research to forensic science and medical diagnostics. This review aims to explore the latest advancements in DNA extraction techniques, highlighting their principles, applications, and suitability for various types of biological samples. Traditional DNA extraction methods, such as phenol-chloroform and ethanol precipitation, have served as the backbone of DNA isolation for decades. However, these techniques often involve hazardous chemicals and can be time-consuming. Recent advancements have focused on developing safer, faster, and more efficient methods, with a focus on automation and scalability. Innovations such as magnetic bead-based extraction, silica column purification, and specialized kits have greatly simplified the process, allowing for high-throughput applications in clinical and research settings. In addition to these technical improvements, new approaches have emerged to address specific challenges, such as extracting DNA from degraded or trace samples, as seen in forensic investigations, or isolating cell-free DNA for non-invasive prenatal testing and cancer diagnostics. Moreover, microfluidic devices and lab-on-a-chip technologies are transforming the landscape by enabling DNA extraction from minute samples with minimal reagent consumption. This review discusses the principles underlying these advanced techniques, their benefits and limitations, and the specific contexts in which they excel. It also considers future trends, including further automation, integration with sequencing platforms, and the potential for point-of-care applications. By examining the current state of DNA extraction technology, this review aims to provide researchers and practitioners with a comprehensive guide to the best methods for their specific needs.

DNA Extraction Methods

KATIE LONG and MARK FLOOD, Forensic Science Program, Fairmont State University, Fairmont, WV 26554. Evaluation of DNA Extraction Methods. Three forms of DNA extraction types include solution-based, column-based, and magnetic bead-based methodologies, with the latter being the accepted methodology used in forensic laboratories. Magnetic bead-based DNA extraction uses the negative charge of DNA and the positive charge of the magnetic beads to isolate the DNA strands and produce a purified sample. However, solution and column-based extraction methods use the same techniques of washing to purify DNA and typically have comparable DNA yields. Cheek swabs were obtained from ten individuals and the three extraction methods were performed to determine the quantitation of double-stranded DNA and the quality of the gel electrophoresis profile. Preliminary results indicate that solution-based extraction produces a slightly higher yield of double-stranded DNA having a 3.92% difference between the average quantitation of 26.29 ng/µL from solution-based and 25.28 ng/µL from magnetic bead-based methods. However, it is expected that the magnetic bead-based extraction will produce higher quality gel electrophoresis results. Overall, the methods examined produced similar DNA extraction efficiencies. This research was made possible by NASA West Virginia Space Grant Consortium, Training Grant #80NSSC20M0055 and the STaR Sure Grant.

A novel, magnetic bead-based extraction method for the isolation of antimicrobial resistance genes with a case study in river water in Malawi.

The environment is increasingly recognized as an important reservoir of antimicrobial resistance genes (ARGs), which can be identified using molecular platforms. Yet, environmental surveillance remains an underutilised tool as there is no agreement on the best strategy for sample processing. We aim to develop a low-cost extraction method independent to commercial kits or reagents. We present a novel, magnetic bead-based method for the isolation of ARGs from river water named MagnaExtract. We present this with analytic limit of detection as well as a case study in Southern Malawi. Here we compare the DNA yield from MagnaExtract with commercially available QIAGEN kits and the crude boil and spin method, using a high-resolution melt analysis PCR panel designed for the detection of third-generation cephalosporin and carbapenem-resistant genes from 98 water samples. The MagnaExtract method is comparable, and in some instance's superior to commercially available kits for the isolation of ARGs from river water samples. The MagnaExtract approach offers a simple, affordable, high yielding extraction method that could be used for the detection of ARGs from river water samples in surveillance campaigns in East Africa.

A microfluidic platform for high-purity cell free DNA extraction from plasma for non-invasive prenatal testing.

Increase the yield and purity of cell-free DNA (cfDNA) extracted from plasma for non-invasive prenatal testing (NIPT) as inefficiencies in this extraction and purification can dramatically affect the sensitivity and specificity of the test. This work integrates cfDNA extraction from plasma with a microfluidic chip platform by combining magnetic bead-based extraction and electroosmotic flow on the microfluidic chip. Various wash buffers and voltage conditions were simulated using COMSOL Multiphysics Modeling and tested experimentally. When performing the first wash step of this assay on the microfluidic chip with 300V applied across the channel there was a six-fold increase in the A260 /A230 ratio showing a significant improvement (p value 0.0005) in the purity of the extracted sample all while maintaining a yield of 68.19%. These values are critical as a high yield results in more sample to analyze and an increase in A260 /A230 ratio corresponds to a decrease in salt contaminants such as guanidinium thiocyanate which can interfere with downstream processes during DNA library preparation and potentially hinder the NIPT screening results. This technique has the potential to improve NIPT outcomes and other clinically relevant workflows that use cfDNA as an analyte such as cancer detection.

High-Throughput CRISPR-Cas13 SARS-CoV-2 Test.

The ability to control the spread of COVID-19 continues to be hampered by a lack of rapid, scalable, and easily deployable diagnostic solutions. We developed a diagnostic method based on CRISPR (clustered regularly interspaced short palindromic repeats) that can deliver sensitive, specific, and high-throughput detection of Sudden Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). The assay utilizes SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the qualitative detection of SARS-CoV-2 RNA and may be performed directly on a swab or saliva sample without nucleic acid extraction. The assay uses a 384-well format and provides results in <1 hour. Assay performance was evaluated with 105 (55 negative, 50 positive) remnant SARS-CoV-2 specimens previously tested using Food and Drug Administration emergency use authorized assays and retested with a modified version of the Centers for Disease Control and Prevention (CDC) quantitative PCR with reverse transcription (RT-qPCR) assay. When combined with magnetic bead-based extraction, the high-throughput SHERLOCK SARS-CoV-2 assay was 100% concordant (n = 60) with the CDC RT-qPCR. When used with direct sample addition the high-throughput assay was also 100% concordant with the CDC RT-qPCR direct method (n = 45). With direct saliva sample addition, the negative and positive percentage agreements were 100% (15/15, 95% CI: 81.8-100%) and 88% (15/17, 95% CI: 63.6-98.5%), respectively, compared with results from a collaborating clinical laboratory. This high-throughput assay identifies SARS-CoV-2 from patient samples with or without nucleic acid extraction with high concordance to RT-qPCR methods. This test enables high complexity laboratories to rapidly increase their testing capacities with simple equipment.

Facile Coupling of Droplet Magnetofluidic-Enabled Automated Sample Preparation for Digital Nucleic Acid Amplification Testing and Analysis

Digital nucleic acid amplification testing (dNAAT) and analysis techniques, such as digital polymerase chain reaction (PCR), have become useful clinical diagnostic tools. However, nucleic acid (NA) sample preparation preceding dNAAT is generally laborious and performed manually, thus creating the need for a simple sample preparation technique and a facile coupling strategy for dNAAT. Therefore, we demonstrate a simple workflow which automates magnetic bead-based extraction of NAs with a one-step transfer to dNAAT. Specifically, we leverage droplet magnetofluidics (DM) to automate the movement of magnetic beads between small volumes of reagents commonly employed for NA extraction and purification. Importantly, the buffer typically used to elute the NAs off the magnetic beads is replaced by a carefully selected PCR solution, enabling direct transfer from sample preparation to dNAAT. Moreover, we demonstrate the potential for multiplexing using a digital high-resolution melt (dHRM) after the digital PCR (dPCR). The utility of this workflow is demonstrated with duplexed detection of bacteria in a sample imitating a coinfection. We first purify the bacterial DNA into a PCR solution using our DM-based sample preparation. We then transfer the purified bacterial DNA to our microfluidic nanoarray to amplify 16S rRNA using dPCR and then perform dHRM to identify the two bacterial species.

Evaluation of beta-thalassemia in the fetus through cffDNA with multiple polymorphisms as a haplotype in the beta-globin gene.

Invasive biopsy during the pregnancy is associated with an abortion risk of approximately 1% for the fetus. Free fetal DNA in maternal plasma is an excellent source of genetic material for prenatal molecular diagnoses. This study was conducted to investigate beta-thalassemia mutation in the fetus through maternal blood with multiple polymorphisms as haplotypes in the beta-globin gene. In this study, a total of 33 beta-thalassemia carrier (minor) couples were genotyped by ARMS-PCR for IVSII-IG>A mutation. During pregnancy, 10mL of blood was collected from pregnant women, and DNA was extracted by the magnetic bead-based extraction, and fetal DNA was enriched with AMPure XP kit. Five polymorphisms in 4 haplotype groups were evaluated by the Sanger Sequencing method. Finally, results were compared with those of the invasion method. Participants in study were 33 couples, mean age of the men was 26±5 years, and mean age of women was 23±4 years, and mean MCV, MCH, HbA2 blood parameters were 62.4±5.3, 19.6±3.1, 4.2±2.1 respectively. A total of 33 fetuses were genotyped for IVSII-IG>A mutation. Nine fetuses were affected, 10 fetuses were normal and 14 fetuses were carrier of beta-thalassemia. Sensitivity and specificity of Sanger Sequencing were equal to 88.8% and 91.6% respectively. Positive and negative predictive values were obtained as 80% and 95.6%, respectively. Mutational status of the fetus can be assessed by determining inheritance of paternally-derived alleles based on detection of haplotype-associated SNP in maternal plasma. Magnetic-based DNA extraction and fetal DNA enrichment are very simple and easy to perform and have satisfactory accuracy.

Comparison of methods for the isolation of cell-free DNA from cell culture supernatant.

In vitro characterization of cell-free DNA using two-dimensional cell culture models is emerging as an important step toward an improved understanding of the physical and biological characteristics of cell-free DNA in human biology. However, precise measurement of the cell-free DNA in cell culture medium is highly dependent on the efficacy of the method used for DNA purification, and is often a juncture of experimental confusion. Therefore, in this study, we compared six commercially available cell-free DNA isolation kits for the recovery of cell-free DNA from the cell culture supernatant of a human bone cancer cell line (143B), including two magnetic bead-based manual kits, one automated magnetic bead-based extraction method, and three manual spin-column kits. Based on cell-free DNA quantitation and sizing, using the Qubit dsDNA HS assay and Bioanalyzer HS DNA assay, respectively, the different methods showed significant variability concerning recovery, reproducibility, and size discrimination. These findings highlight the importance of selecting a cell-free DNA extraction method that is appropriate for the aims of a study. For example, mutational analysis of cell-free DNA may be enhanced by a method that favors a high yield or is biased toward the isolation of short cell-free DNA fragments. In contrast, quantitative analysis of cell-free DNA in a comparative setting (e.g. measuring the fluctuation of cell-free DNA levels over time) may require the selection of a cell-free DNA isolation method that forgoes a high recovery for high reproducibility and minimal size bias.

Direct Magnetic Bead-Based Extraction of MicroRNA from Urine with Capillary Electrophoretic Analysis Using Fluorescence Detection and Universal Label.

Short non-coding RNAs, specifically microRNAs (miRNAs), are of a great interest due to their presumed function in genome regulation. Moreover, miRNAs are currently perceived as potential biomarkers for numerous diseases; a variety of detection methods and sensing systems have therefore been studied. We present a magnetic-bead-based assay for specific miRNA isolation coupled with sensitive electrophoretic analysis with fluorescence detection. The magnetic separation step involves creating a duplex with targeted miR-141, which is subsequently cleaved from the magnetic bead surface with a specific endonuclease. The duplex is then determined using capillary electrophoresis with laser-induced fluorescence detection in the presence of the fluorescent dye PicoGreen for quantitating double-stranded DNA. The benefits of using microcolumn separation technique coupled with sensitive detection over traditionally used determination by fluorescence spectrometry include the fact that there is no need for a specific pre-labeled fluorescent probe. This significantly simplifies the method and reduces the costs. Cross-reactivity with mismatched oligonucleotides (3 and 5 mismatched bases) and different miRNAs (miR-124 and miR-150) was tested, demonstrating the specificity of the developed method for miRNA-141. This magnetic extraction method was demonstrated for the direct isolation and determination of miR-141 at different concentration levels from urine samples and the achieved nanomolar detection limit.

Touch DNA sampling with SceneSafe Fast™ minitapes

To achieve optimal results in the forensic analysis of trace DNA, choosing the right collection technique is crucial. Three common approaches are currently well-established for DNA retrieval from items of clothing, notably cutting, swabbing and tape-lifting. The latter two are non-destructive and therefore preferable on items of value. Even though the most recently established technique of DNA retrieval by adhesive tapes is widely used since quite some years now, little information has been published so far on how well it performs compared to other methods. Even more important, when it comes to choosing the right DNA extraction method for forensic lifting-tapes, the available information one can rely on as a forensic geneticist is quite scarce. In our study we compared the two widely used, commercially available and automation suitable magnetic bead-based extraction methods "iPrep Forensic Kit" and "PrepFiler Express BTA™ Kit" to conventional organic solvent extraction. The results demonstrate that DNA extraction from standardized saliva samples applied to SceneSafe Fast™ minitapes is most efficient with phenol-chloroform. We also provide evidence that SceneSafe Fast™ minitapes perform better than wet cotton swabs in the sampling of touch DNA from cotton fabric. Applying the tape only once in every spot on the tissue is thereby sufficient for a considerably better collection performance of the tapes compared to swabbing.

The research and application of pretreatment method for matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of filamentous fungi

Objective: To establish and evaluate the feasibility of a pretreatment method for matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of filamentous fungi developed by the laboratory. Methods: Three hundred and eighty strains of filamentous fungi from January 2014 to December 2016 were recovered and cultured on sabouraud dextrose agar (SDA) plate at 28 ℃ to mature state. Meanwhile, the fungi were cultured in liquid sabouraud medium with a vertical rotation method recommended by Bruker and a horizontal vibration method developed by the laboratory until adequate amount of colonies were observed. For the strains cultured with the three methods, protein was extracted with modified magnetic bead-based extraction method for mass spectrum identification. Results: For 380 fungi strains, it took 3-10 d to culture with SDA culture method, and the ratio of identification of the species and genus was 47% and 81%, respectively; it took 5-7 d to culture with vertical rotation method, and the ratio of identification of the species and genus was 76% and 94%, respectively; it took 1-2 d to culture with horizontal vibration method, and the ratio of identification of the species and genus was 96% and 99%, respectively. For the comparison between horizontal vibration method and SDA culture method comparison, the difference was statistically significant (χ(2)=39.026, P<0.01); for the comparison between horizontal vibration method and vertical rotation method recommended by Bruker, the difference was statistically significant(χ(2)=11.310, P<0.01). Conclusion: The horizontal vibration method and modified magnetic bead-based extraction method developed by the laboratory is superior to the method recommended by Bruker and SDA culture method in terms of the identification capacity for filamentous fungi, which can be applied in clinic.

Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis.

Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95–100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting.

Magnetic bead technology for viral RNA extraction from serum in blood bank screening

Nucleic acid amplification testing (NAT) was recently recommended by Brazilian legislation and has been implemented at some blood banks in the city of São Paulo, Brazil, in an attempt to reduce blood-born transmission of human immunodeficiency virus (HIV) and hepatitis C virus. ObjectiveManual magnetic particle-based extraction methods for HIV and HCV viral nucleic acids were evaluated in combination with detection by reverse transcriptase -polymerase chain reaction (RT-PCR) one-step. MethodsBlood donor samples were collected from January 2010 to September 2010, and minipools of them were submitted to testing. ELISA was used for the analysis of anti-HCV/HIV antibodies. Detection and amplification of viral RNA was performed using real-time PCR. ResultsOut of 20.808 samples screened, 53 samples (29 for HCV and 24 for HIV) were confirmed as positive by serological and NAT methods. ConclusionThe manual magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donation for viremic donors to further increase the safety of blood products.

Comparative evaluation of two automated systems for nucleic acid extraction of BK virus: NucliSens easyMAG versus BioRobot MDx

The objective of this study was to compare the performance of two automated nucleic acid extraction systems. Specifically, the NucliSens easyMAG system (bioMerieux, Marcy l’Etoile, France), which incorporates magnetic bead technology, was compared with the BioRobot MDx system (Qiagen GmbH, Hilden, Germany), which uses a silica membrane-based method of nucleic acid extraction. Nucleic acids from the BK virus (BKV DNA) were extracted from 98 plasma and 57 urine specimens using the Real-Q BKV quantitation kit (Biosewoom, Seoul, Korea). Failed PCR was defined as negative BKV DNA results having more than 36 threshold cycles of the internal control by the manufacturer's instruction. The PCR failure rate of nucleic acids isolated from plasma samples using the MDx system was similar to that of plasma samples processed using the easyMAG system (2.0% and 3.1%, respectively). The PCR failure rate of nucleic acids isolated from urine samples using the MDx system was higher than that of urine samples processed using the easyMAG system (33.3% and 12.5%, respectively). These data suggest that the PCR inhibitors present in urine specimens are removed more efficiently by the easyMAG system. Among amplified specimens, the discordant results obtained from the two systems revealed that the BKV DNA load ranged from 2.3 log 10 copies/mL to 4.6 log 10 copies/mL. Of the 25 urine specimens that yielded BKV DNA by both extraction systems, 15 specimens (60.0%) yielded higher BKV DNA loads by the easyMAG system, indicating that the easyMAG system extracted nucleic acid more efficiently than did the MDx system. In conclusion, the easyMAG method outperformed the MDx method when used to extract BKV DNA from urine samples. Magnetic bead-based extraction methods such as the easyMAG system are therefore preferable for the quantitation of viral DNA in urine.