Separation Of DNA Fragments Research Articles

Partitioning behavior of short DNA fragments in polymer/salt aqueous two-phase systems.

The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.

Microbial Diversity Assessment in Soil from Dumping Sites: Isolation, DNA Extraction and Electrophoretic Analysis

This study aims to investigate the microbial diversity present in soil samples collected from various dumping sites, which are known for their complex and heterogeneous waste environments. The primary objective is to isolate and characterize the microbial communities inhabiting these soils, providing insights into their composition and potential ecological roles. Soil samples were collected by digging 5 cm deep at multiple dumping locations to ensure a representative sampling of the microbial populations. The collected soil samples underwent a serial dilution process, extending up to 10^-9 and 10^-10 dilutions, to facilitate the isolation of individual microbial colonies. These diluted samples were then inoculated onto Standard Potato Dextrose Agar (PDA) media, a nutrient-rich medium conducive to microbial growth. Following an incubation period, distinct bacterial colonies were observed and isolated for further analysis. DNA was extracted from the isolated bacterial colonies using a standard DNA extraction protocol. The quality and purity of the extracted DNA were assessed through gel electrophoresis, a technique that separates DNA fragments based on their size. Remarkably, the electrophoretic analysis revealed clear and distinct DNA bands along with RNA bands, indicating successful extraction of high-quality microbial DNA. Notably, this was achieved without the use of proteinase treatment, which is typically employed to remove protein contaminants from DNA samples. The results of this study underscore the robustness of the methodologies employed in isolating and characterizing microbial DNA from soil samples collected at dumping sites. The presence of distinct DNA and RNA bands highlights the effectiveness of the DNA extraction process, even in the absence of proteinase treatment. These findings provide valuable insights into the microbial diversity within dumping site soils, suggesting a rich and varied microbial community that may play significant roles in soil health and waste decomposition.

Rhesus Macaques: VNTR Polymorphism of the FCGRT Gene.

Variable number tandem repeat (VNTR) polymorphisms of the human neonatal IgG Fc receptor α-chain gene (FCGRT) are known to influence the expression levels of FCGRT and IgG in serum. Monkeys are considered to be a relevant biological model for studying the effects of immunobiological drugs. The study determined the functional VNTR polymorphisms of the FCGRT gene in 109 male rhesus macaques from the nursery of the Kurchatov Complex of Medical Primatology. PCR amplification of samples was carried out followed by electrophoretic separation of DNA fragments in a 2% agarose gel. Individual DNA amplification products were sequenced (according to Sanger system) in forward and reverse directions to confirm the specificity. The genotyping showed that the VNTR polymorphism of the FCGRT gene in the studied population of rhesus macaques is presented by 9 variants. The frequency of the VNTR5 allele associated with lower IgG levels was 14.2%, and the most common one was the VNTR7 allele (25.2%). We also identified alleles that have not been previously reported: VNTR3, VNTR4, VNTR6, VNTR8, and VNTR9. The study allows to consider rhesus macaques as a potential model for studying the immunological response depending on the genetic VNTR variant of FCGRT.

Nanoconfined Electrokinetic Chromatography (NEC): Gradient Separation and Sensing of Short DNA Fragments at the Single-Molecule Level.

Glass nanopipets have been demonstrated to be a powerful tool for the sensing and discrimination of biomolecules, such as DNA strands with different lengths or configurations. Despite progress made in nanopipet-based sensors, it remains challenging to develop effective strategies that separate and sense in one operation. In this study, we demonstrate an agarose gel-filled nanopipet that enables hyphenated length-dependent separation and electrochemical sensing of short DNA fragments based on the electrokinetic flow of DNA molecules in the nanoconfined channel at the tip of the nanopipet. This nanoconfined electrokinetic chromatography (NEC) method is used to distinguish the mixture of DNA strands without labels, and the ionic current signals measured in real time show that the mixed DNA strands pass through the tip hole in order according to the molecular weight. With NEC, gradient separation and electrochemical measurement of biomolecules can be achieved simultaneously at the single-molecule level, which is further applied for programmable gene delivery into single living cells. Overall, NEC provides a multipurpose platform integrating separation, sensing, single-cell delivery, and manipulation, which may bring new insights into advanced bioapplication.

Application of Molecular Biology Technologies in Environmental restoration

With the development of molecular biology, various new technologies have been developed and applied to the monitoring and management of the ecological environment. This includes the biodiversity of the oceans, soils, and plant and microbial ecosystems. This paper introduces the types of molecular biology techniques and discusses their applications in bioremediation and monitoring of contaminated environments. Conventional techniques include genetic engineering, DNA sequence analysis, electrophoresis separation and purification, nucleic acid probe detection, and polymerase chain reaction. Denaturing gradient gel electrophoresis (denaturing gradient gel electrophoresis) is a new electrophoresis technique. It is a gel system that separates DNA fragments according to their melting properties. Gene microarray technology (also known as gene chip) began to be widely used in the 1890s with biological monitoring technology. There are thousands of nucleic acid probes, which detect nucleic acids in living organisms using fluorescence or electricity, in gene microarray technology. These technologies can be used to regulate the diversity of microbial communities, solve the virus infection of plants, and explore the contaminant degradation genes and functional strains. The development and progress of modern molecular biology technology and biochemical technology have promoted the research and detection of ecological environments. These methods would become very effective tools for the study of environmental ecology.

Emulsion Polymerase Chain Reaction Coupled with Denaturing Gradient Gel Electrophoresis for Microbial Diversity Studies.

Emulsion PCR-DGGE is a molecular biology technique used to amplify and analyze DNA fragments. This technique combines two processes, emulsion PCR and denaturing gradient gel electrophoresis (DGGE), to enhance the specificity and yield of the amplification process and to separate the amplified fragments based on their melting behavior. In the emulsion PCR step, a high-quality DNA template is mixed with the PCR reagents and droplet generator oil to create an oil-in-water emulsion. The emulsion is then subjected to thermal cycling to amplify the target DNA fragments. The amplified fragments are recovered from the droplets and purified to remove any impurities that may interfere with downstream applications. In the DGGE step, the purified amplicon is loaded onto a DGGE apparatus, where the DNA fragments are separated and visualized based on their melting behavior. This method allows for the concurrent amplification and separation of multiple DNA fragments, thereby enhancing the resolution and sensitivity of the analysis. It is widely used in environmental and medical microbiology research, as well as in other fields that require the identification and characterization of microorganisms, such as the study of microbial diversity in soil, water, and other natural environments, as well as in the human gut microbiome and other medical samples.

Genetic Diversity Study of Melon Native Landraces Using ISSR Markers in Kurdistan Region of Iraq

Considering the ancient cultivation of various numbers of melons, it seems that there is a valuable and rich gene pool of melon in Kifri city of Kurdistan region of Iraq. In this research, the genetic diversity of 15 indigenous melons of Kifri and 7 non-native melons was analyzed using inter simple sequence repeat (ISSR) markers. A total of 6 primers were used to amplify parts of the genomic DNA of the samples. Genomic DNA was extracted using Salting out method, and polyacrylamide gel was used to separate DNA fragments. In this study, 65 bands were scored and 34 bands had polymorphisms. To evaluate the genetic similarity between the samples, cluster analysis was used using the Jaccard similarity coefficient with the UPGMA method. The average genetic distance between the samples using the Jaccard similarity coefficient was 0.74, and the average polymorphic information content (PIC) was 0.78. The primer (AC)8 G had the highest PIC (0.93). Cluster analysis indicated wide range of diversity across the genotypes used. These genotypes have high diversity which could be used in breeding programs.

Dual Catalytic Hairpin Assembly-Based Automatic Molecule Machine for Amplified Detection of Auxin Response Factor-Targeted MicroRNA-160.

MicroRNA160 plays a crucial role in plant development by negatively regulating the auxin response factors (ARFs). In this manuscript, we design an automatic molecule machine (AMM) based on the dual catalytic hairpin assembly (D-CHA) strategy for the signal amplification detection of miRNA160. The detection system contains four hairpin-shaped DNA probes (HP1, HP2, HP3, and HP4). For HP1, the loop is designed to be complementary to miRNA160. A fragment of DNA with the same sequences as miRNA160 is separated into two pieces that are connected at the 3′ end of HP2 and 5′ end of HP3, respectively. In the presence of the target, four HPs are successively dissolved by the first catalytic hairpin assembly (CHA1), forming a four-way DNA junction (F-DJ) that enables the rearrangement of separated DNA fragments at the end of HP2 and HP3 and serving as an integrated target analogue for initiating the second CHA reaction, generating an enhanced fluorescence signal. Assay experiments demonstrate that D-CHA has a better performance compared with traditional CHA, achieving the detection limit as low as 10 pM for miRNA160 as deduced from its corresponding DNA surrogates. Moreover, non-target miRNAs, as well as single-base mutation targets, can be detected. Overall, the D-CHA strategy provides a competitive method for plant miRNAs detection.

First experiences with the Spectrum Compact CE System

The newly released Spectrum Compact CE System by Promega is a capillary electrophoresis instrument developed for DNA-fragment separation and sequencing. In this study, its compatibility to 8 commercial short tandem repeat (STR) kits from 4 different manufacturers, reproducibility (sizing precision, accuracy and concordance) and robustness (sensitivity and mixture resolution) were tested and compared to the ABI PRISM® 310 Genetic Analyzer. The instrument was able to successfully analyse amplicons of all tested kits, proved to be as precise as claimed by manufacturer specifications and was shown to be more robust than the ABI PRISM® 310 Genetic Analyzer in some aspects. Analyses on the Spectrum Compact CE System were able to resolve peaks with length differences of 1-basepair in the short and long fragment range and mixtures of mixture ratios down to 1:30. We describe the advantages and limitations we have observed so far working with this instrument in our forensic genetics laboratory.

Detection and Separation of Single-Stranded DNA Fragments Using Solid-State Nanopores.

Many biological assays require effectively and sensitively sorting DNA fragments. Here, we demonstrate a solid-state nanopore platform for label-free detection and separation of short single-stranded DNA (ssDNA) fragments (<100 nt), based on their length-dependent translocation behaviors. Our experimental data show that each sized pore has a passable length threshold. The negative charged ssDNA fragments with length smaller than the threshold can be electrically facilitated driven through the correspondingly sized nanopore along the direction of electric field. In addition, the passable length threshold increases with the pore size enlarging. As a result, this phenomenon is able to be applicable for the controllable selectivity of ssDNA by tuning nanopore size, and the selectivity limitation is up to 30nt. Numerical simulation results indicate the translocation direction of ssDNA is governed by the competition of electroosmosis and electrophoresis effects on the ssDNA and offer the relationship between passable length threshold and pore size.

SELECTION AND ASSESSMENT OF ISSR-MARKERS FOR ANALYSIS OF SEPARATE CATTLE POPULATIONS

The article covers molecular genetic analysis of intermicrosatellite DNA sequences of dairy cattle productivity. Molecular markers based on DNA polymorphism were used for this monitoring. Such markers make it possible to assess quickly the genetic polymorphism of taprin in the herd. Іnsofar as a large number of intermicrosatellite repeats is in the genome of cattle, that increases the probability of detecting polymorphic loci. The ISSR markers selected for the study are based on multiclocus synthesis in polymerase chain reaction (PCR) and allow an objective study of the breed and interbreed diversity. And it makes possible to assess quickly and accurately genetic diversity for the presence of genes associated with economically useful traits. The purpose of this work is the selection and evaluation of ISSR-markers for the analysis and study of genetic diversity of Ukrainian and imported breeds of dairy cattle. Samples of biological material from representatives of three herds of cattle (Ukrainian Red-and-White spotted dairy, Montbeliard breed and their crossbreeds) were selected for the study by the method of groups-analogues. For the analysis of this material the generally accepted zootechnical methods of studying of a selection material and methods of an estimation of animals on molecular – genetic markers are included. According to standard methods, DNA was isolated from peripheral blood lymphocytes using a set of reagents "DNA Sorb B". Amplification of total DNA with ISSR primers was performed on a thermal cycler "Tertsyk" ("DNA technology" of the Russian Federation). Electrophoretic separation of DNA fragments was performed in an agarose gel according to conventional methods. The size of the obtained PCR products was detected using a molecular weight marker SM1343. As a result of the study of the biological material of these animals, the obtained ISSR-PCR products were quite heterogeneous. The vast majority of polylocus spectra had clear discrete bands, but there were amplicons without clear discrete bands. Analyzing the results of the study of the genetic structure of animals of the Ukrainian Red-and-White dairy breed, using primers ISSR-1, ISSR-2, ISSR-3 and ISSR-4, the range of obtained PCR products ranges from 250 bp. up to 3000 bp. The range of amplification products in Montbeliard animals has a smaller range and ranges from 250 bp, respectively. up to 1500 bp.The obtained amplicons for the use of primers ISSR-1 and ISSR-2, ISSR-3 and ISSR-4 in the turf of Ukrainian Red-and-White dairy and Montbeliard breeds have sizes from 350 bp to 2000 bp. Having analyzed the information you can determine the distribution of the number and length of DNA fragments using 4 ISSR-markers. The total number of amplified DNA fragments varies depending on the primer from 21 to 106, and their size ranges from 250 BP up to 3000 bp. Based on the analysis of DNA plymorphism, it is possible to assess the heterogeneity of selected populations of cattle. Thus, as a result of studying the genetic structure of animals of two breeds of dairy cattle and their crossbreeds by intermicrosatellite DNA loci, their individual polymorphism was revealed. The amplification products have significant variations depending on the primer used. Primers ISSR-1 and ISSR-2 were the most informative for the analysis of cattle DNA polymorphism.

Effect of Electric Field Voltage on the Efficiency of Separation of Single-Stranded DNA Fragments by Capillary Gel Electrophoresis in Polymers Based on Linear Poly(N,N-Dimethylacrylamide)

The analysis of DNA fragments by capillary gel electrophoresis is in wide current used in the practice of research and testing laboratories primarily for highly accurate and highly sensitive identification of organisms by their DNA traces separated from biological test materials and amplified by polymerase chain reaction. A constantly growing flow of examinations performed using this method necessitates the optimization of research protocols in order to shorten the analysis time without loss of separation efficiency. The effect of electric field voltage on the separation efficiency of single-stranded DNA fragments in a length range from 60 to 1200 nucleotides in a series of polymers based on linear poly(N,N-dimethylacrylamide) was studied. The best values of electric field voltage were experimentally determined to be recommended for DNA fragment analysis on the first Russian genetic analyzer Nanophore 05 with the studied polymers.

Comparative Analysis of ANDE 6C Rapid DNA Analysis System and Traditional Methods.

Rapid DNA analysis is an ultrafast and fully automated DNA-typing system, which can produce interpretable genetic profiles from biological samples within 90 minutes. This “swab in—profile out” method comprises DNA extraction, amplification by PCR multiplex, separation and detection of DNA fragments by capillary electrophoresis. The aim of study was the validation of the Accelerated Nuclear DNA Equipment (ANDE) 6C system as a typing method for reference samples according to the ISO/IEC 17025 standard. Here, we report the evaluation of the validity and reproducibility of results by the comparison of the genetic profiles generated by the ANDE 6C System with those generated by standard technologies. A quantity of 104 buccal swabs were analyzed both through the ANDE 6C technology and the traditional method (DNA extraction and quantification, amplification and separation by capillary electrophoresis). Positive typing was observed in 97% of cases for ANDE 6C technology with only three buccal swabs failing to reveal interpretable signals. Concordance was determined by comparing the allele calls generated by ANDE 6C and conventional technology. Comparison of 2800 genotypes revealed a concordance rate of 99.96%. These results met the ISO/IEC 17025 requirements, enabling us to receive the accreditation for this method. Finally, rapid technology has certainly reached a level of reliability which has made its use in laboratories of forensic genetics a reality.

Abstract 3529: Sample quality control of cell-free DNA

Abstract Sequencing of cell-free DNA (cfDNA) is possible due to the establishment of low input library protocols for next-generation sequencing workflows. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mononucleosome with a size around 170 bp, sometimes with additional species representing nucleosome multimers. Further, cfDNA samples may contain larger DNA fragments dependent on pre-analytical sample treatment or extraction method. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. Therefore, reliable quantification of cfDNA requires a method that separates DNA fragments by size, such as electrophoresis. This poster shows the use of an automated electrophoresis platform performing cfDNA quantification with region analysis. Moreover, the results include a score to qualify cfDNA samples according to their contamination level with high molecular weight material. This allows defining a threshold for objective sample qualification prior to library preparation. The analysis features are described with examples of typical sample patterns. Citation Format: Eva Graf. Sample quality control of cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3529.

Study of the peak broadening due to detection in the electrophoretic separation of DNA by CE and microchip CE and the application of image sensor for ultra-small detection cell length.

Narrow peaks are important to high-resolution and high-speed separation of DNA fragments by capillary electrophoresis and microchip capillary electrophoresis. Detection cell length is one of the broadening factors, which is often ignored in experiments. However, is it always safe to neglect detection cell length under any condition? To answer this question, we investigated the influence of detection cell length by simulation and experiments. A parameter named as detection cell length ratio was proposed to directly compare the detection cell length and the spatial length of sample band. Electrophoretic peaks generated by various detection cell length ratios were analyzed. A simple rule to evaluate the peak broadening due to detection cell length was obtained. The current states of the detection cell length of detection system and their reliabilities in capillary electrophoresis and microchip capillary electrophoresis were analyzed. Microchip capillary electrophoresis detection with an ultra-small detection cell length of 0.36μm was easily achieved by using an image sensor.

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes.

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Abstract 5587: Sample quality control of cell-free DNA

Abstract Sequencing of cell-free DNA (cfDNA) extracted from blood specimens or other body fluids is possible due to the establishment of low input library protocols for next-generation sequencing workflows. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mononucleosome with a size around 170 bp, sometimes with additional species representing nucleosome multimers. Further, cfDNA samples may contain larger DNA fragments dependent on the donor's health status, preanalytical sample treatment, or extraction method. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. Therefore, reliable quantification of cfDNA requires a method that separates DNA fragments by size, such as electrophoresis. This poster shows the use of a new cfDNA assay for the 2100 Bioanalyzer system as an add-on for the High Sensitivity DNA assay. The assay enables automated cfDNA quantification with pre-defined regions. Moreover, the results include sample purity as a score to qualify cfDNA samples according to their contamination level with high molecular weight material. The features of the assay are described with examples of typical sample patterns. Citation Format: Melissa H. Liu, Eva Graf. Sample quality control of cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5587.

The effect of electrophoretic parameters on separation performance of short DNA fragments

Effective separation of short DNA fragments is important for the identification of PCR or LAMP products. We investigated the effect of electric field strength, sample plug width, effective length of the capillary, concentration and molecular weight of polymer on the separation performance of small DNA. Results demonstrated that the sample plug played a non-negligible role in the peak broadening. The migration time of DNA was exponentially decreased with the increase of electric field strength. Increasing effective length of capillary, concentration or molecular weight of HEC may improve the separation performance, but it was at the cost of long migration time.

Influence of the Sample Volume upon Injection on Capillary Gel Electrophoresis for the Separation of Small DNA Fragments

Influence of the Sample Volume upon Injection on Capillary Gel Electrophoresis for the Separation of Small DNA Fragments

Ultraaccurate genome sequencing and haplotyping of single human cells

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10-8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs.