DNA Capillary Electrophoresis Research Articles

Validation of a Forensic Method to be used in Food Fraud Investigation

Food fraud is the act in which food is purposefully mislabeled, adulterated, or contaminated, often to make up a percentage of a missing substance when the product is being made. It is often done purposefully to mislead consumers into thinking some ingredient is or is not present in the food, which can be hazardous to the consumer. This project aimed to validate a forensic domestic animal species reference ladder and multiplex PCR kit that can be used in animal investigations, with a focus on food fraud. A multiplex PCR protocol targeting mitochondrial DNA (mtDNA) markers and a species reference ladder can quickly identify DNA from chicken, cow, sheep, turkey, goat, horse, dog, and cat. To validate the kit, it was tested on five different brands of dog food, specifically four brands of kibble alongside a canned wet stew dog food. DNA extraction was performed using the QIAamp MinElute Column kit or the Qiagen EZ1 investigator kit, then DNA quantification, multiplex PCR, gel electrophoresis, and capillary electrophoresis to build species mtDNA profiles for each food’s contents. Results from one dog food kibble yielded some speculation as DNA for sheep and goat contents not listed in the ingredients were detected using both the gel and capillary electrophoresis.

Combination of upstream primer-multuplex PCR (UP-mpcr) and capillary electrophoresis for equine genetic analysis

The purity and content of DNA extracted from the sample is important during PCR analysis. In the conditions of our country, there are many cases of working on samples that do not meet the requirements for some reason. In such cases, there is a need to further test and develop other sensitive methods. The upstream-primer multiplex PCR (UP-mPCR technique) is known for its high specificity and fidelity, and has been used for detecting multiple food borne pathogens, meat species testing and detecting different genetically modified organism (GMO) insertions in a plant genome. The purpose of this experiment is to apply the UP-mPCR method on DNA samples that do not meet the quality requirements, and to test it on domestically produced diagnostics.We combined UP-mPCR with fragment analysis on DNA capillary electrophoresis genetic analyzer by applying fluorescent labelled upstream primers which were tested by amplifying 8 STRs on 23 low-quality equine gDNA samples. These samples had formerly undergone unsuccessful testing by domestic equine genotyping 15-plex kit. Single trial of UP-mPCR on the same samples showed successful amplification and detection of amplicons from 4-6 STRs, and their alleles were genotyped. Combining UP-mPCR and DNA capillary electrophoresis can be helpful in extreme situations such as having limited amounts of sample, or a shortage of multiple fluorescent dye oligonucleotides. There is no former report about the same method as combining UP-mPCR with fragment analysis.

Alterations in thymocyte populations under conditions of endotoxin tolerance.

Background:Endotoxin tolerance (ET) is a protective phenomenon in which pre-treatment with a tolerance dose of lipopolysaccharide (LPS) leads to dramatically elevated survival. Accumulating evidence has shown that peripheral T cells contribute to the induction of ET. However, what happens to T cell development in the thymus under ET conditions remains unclear. The purpose of this study was to analyze the alterations in thymocyte populations (double-positive [DP] and single-positive [SP] cells) under ET conditions.Methods:Mice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups: a group examined 72 h after LPS injection (72-h group) and a group examined 8 days after LPS injection (8-day group). Injection of phosphate-buffered saline was used as a control (control group). Changes in thymus weight, cell counts, and morphology were detected in the three groups. Moreover, surface molecules such as CD4, CD8, CD44, CD69, and CD62L were analyzed using flow cytometry. Furthermore, proliferation, apoptosis, cytokine production, and extracellular signal-regulated kinase (ERK) pathway signaling were analyzed in thymocyte populations. The polymorphism and length of the T-cell receptor (TCR) β chain complementarity-determining region 3 (CDR3) were analyzed using capillary electrophoresis DNA laser scanning analysis (ABI 3730).Results:Thymus weight and cell counts were decreased in the early stage but recovered by the late stage in a murine model of LPS-induced ET. Moreover, the proportions of DP cells (control: 72.130 ± 4.074, 72-h: 10.600 ± 3.517, 8-day: 84.770 ± 2.228), CD4+ SP cells (control: 15.770 ± 4.419, 72-h: 44.670 ± 3.089, 8-day: 6.367 ± 0.513), and CD8+ SP cells (control: 7.000 ± 1.916, 72-h: 34.030 ± 3.850, 8-day: 5.133 ± 0.647) were obviously different at different stages of ET. The polymorphism and length of TCR β chain CDR3 also changed obviously, indicating the occurrence of TCR rearrangement and thymocyte diversification. Further analysis showed that the expression of surface molecules, including CD44, CD69, and CD62L, on thymocyte populations (DP and SP cells) were changed to different degrees. Finally, the proliferation, apoptosis, cytokine production, and ERK pathway signaling of thymocyte populations were changed significantly.Conclusion:These data reveal that alterations in thymocyte populations might contribute to the establishment of ET.

Single Nucleotide Polymorphisms of BMP2 Gene Association with Skeletal Class I Crowding: A PCR Study

This study is conducted to find the association of BMP2 (bone morphogenic protein 2) gene variant rs1005464 and rs15705 with skeletal class I crowding cases. Blood samples from 60 subjects who visited the Department of Orthodontics and Dentofacial Orthopaedics, D.A.P.M.R.V. Dental College, Bengaluru, were taken after written informed consent. These were divided into two groups: group A with 30 subjects having skeletal class I bases with crowding and group B with 30 subjects having skeletal class I bases without visible crowding or spacing (±2 mm). Around 2 mL of venous blood sample was procured from cases and controls after careful examination. All the samples were then subjected to polymerase chain reaction followed by DNA sequencing and capillary electrophoresis. BMP2 rs1005464 and rs15705 gene variants were assessed and Z-Test was used for statistical analysis. GG (p = 0.001) and CC (p = 0.0024) genotype of BMP2 gene variant rs1005464 and rs15705, respectively, are significantly associated with skeletal class I crowding cases. This study concludes that BMP2 variants rs1005464 and rs15705 can be used as genetic markers for skeletal class I bases having crowding. Predisposing genetic markers BMP2 can be identified prior and this would help in predicting the probability of potential crowding in the future and this would help in early prevention and intervention of crowding.

Evaluation of Rapid DNA system for buccal swab and disaster victim identification samples.

An evaluation of a Rapid DNA system was performed using buccal swab samples and mock Disaster Victim Identification (DVI) samples collected postmortem. The allelic ladder success rate was 90% and samples analyzed simultaneously with this allelic ladder were used for further analysis. Sample success rate of the Rapid DNA system for buccal swab samples, and blood and muscle DVI samples were calculated. Success rates of buccal swab samples were 100% and 75% using cassettes preloaded with all reagents suitable for high- and low-DNA content samples, respectively. Success rates of fresh DVI samples were 80% to 100%. Success rates of putrefied DVI samples varied widely between 0% and 20% and 50% to 80% depending on cassette and sample types. Conventional DNA analysis was performed for comparison with the results of the Rapid DNA system. DNA quantity and degradation of human DNA were measured using quantitative polymerase chain reaction. DVI samples that yielded more than 1ng/µL of DNA when extracted with conventional protocols were suitable for analysis using cassettes for both high- and low-DNA content samples. DVI samples with less than 0.1ng/µL of DNA were suitable only for analysis using cassettes for low-DNA content samples. All alleles called and exported by the Expert system software implemented in the Rapid DNA system were concordant with allele calls made by conventional capillary electrophoresis DNA analysis.

Development of methods for effective identification of CRISPR/Cas9-induced indels in rice.

Two methods, PCR and amplicon labeling based, were developed and successfully applied to reliably detect CRISPR/Cas9 induced indels in rice. The use of CRISPR/Cas9 has emerged as a powerful nuclease-based genome editing tool in several model organisms including plants for mutagenesis by inducing precise gene editing through efficient double strand DNA breaks (DSBs) at the target site and subsequent error-prone non-homologous end joining (NHEJ) repair, leading to indel mutations. Different molecular methods including enzymatic mismatch cleavage (EMC), high-resolution melting curve analysis (HRMA) and conventional polymerase chain reaction (PCR) combined with ligation detection reaction (LDR) have been developed to quick identify CRISPR/Cas9 induced mutations. However, their intrinsic drawbacks limit their application in the identification of indel mutants in plants. Here we present two methods (one simple PCR based and the other amplicon labeling based) for effective and sensitive detection of CRISPR/Cas9 induced indels in rice. In PCR-based method, targets were amplified using two pairs of primers for each target locus and visualized on gel electrophoresis, while in amplicon labeling-based method, targets were amplified using tri-primers (with one a universal 6-FAM 5'-labelled) and detected by DNA capillary electrophoresis. Both methods can accurately define indel sizes down to ± 1bp, and are amenable for high throughput analysis, therefore, will significantly facilitate the identification of indel mutants generated by CRISPR/Cas9 for further functional analysis and breeding in rice and other plants.

Structure of Manila Clam (Ruditapes philippinarum) Microbiota at the Organ Scale in Contrasting Sets of Individuals

Marine invertebrate microbiota has a key function in host physiology and health. To date, knowledge about bivalve microbiota is poorly documented except public health concerns. This study used a molecular approach to characterize the microbiota associated with the bivalve Manila clam (Ruditapes philippinarum) by determining (1) the difference among organs either or not under the influence of host habitat, (2) small-scale variability of microbiota, and (3) the experimental response of the Manila clam microbiota submitted to different lateral transmissions. These questions were investigated by sampling two groups of individuals living in contrasting habitats and carrying out a transplant experiment. Manila clam microbiota (i.e., bacterial community structure) was determined at organ-scale (gills, gut, and a pool of remaining tissues) by capillary electrophoresis DNA fingerprinting (CE fingerprinting). The Manila clam microbiota structure differed among organs indicating a selection of Manila clam microbiota at organ scale. Habitat strongly influenced gill and gut microbiota. In contrast, microbiota associated with remaining tissues was similar between group individuals suggesting that these communities are mostly autochthonous, i.e., Manila clam specific. Transplant experiment showed that improving living condition did not induce any change in microbiota associated with remaining tissues. In contrast, the reduction in individual habitat quality led to individuals in declining health as strongly suggested by the increase in phagocytosis activity and decrease in condition index together with the change in internal organ microbiota. This study provides a first description of the Manila clam holobiont which can withstand disturbance and respond opportunistically to improved environmental conditions.

Fast and sensitive detection of indels induced by precise gene targeting.

The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.

IDH1 Splicing Alterations in Patients with AML and Their Relationship to Blood 2HG Levels

Introduction: Point mutations in the human cytosolic isocitrate dehydrogenase 1 (IDH1) gene, or its mitochondrial homolog IDH2 gene, are frequent in patients with AML, particularly at residues R132 in IDH1 and, R140 or R172 in IDH2. These mutations cause loss of the enzymeÕs ability to catalyze the conversion of isocitrate to α-ketoglutarate and result in neomorphic production of R(-)-2-hydroxyglutarate (2HG). It has been reported that 2HG functions as an ÒoncometaboliteÓ. Interestingly, serum levels of 2HG are highly variable among patients with the same mutations, and occasionally are elevated even in patients without detectable mutations. Previously Chaturvedi et al (ASH 2011, Abstract) showed that an IDH1 splice variant promotes leukemogenesis in vivo in the absence of IDH1/IDH2 mutations. In this study, we examined the frequency of this splice variant and two other IDH1 novel splice variants in patients with AML, and looked for any relationships between splice variants, mutations, and 2HG levels.Methods: Cloning and sequencing analysis were used to identify IDH1 and IDH2 splice variants. DNA fragment analysis and capillary electrophoresis was used to evaluate expression of splice variants in 242 patients, of which 166 patients had 2HG blood levels measured. As controls we used two CD34+ bone marrow (BM) cells, three un-fractionated BM cells, and purified neutrophil and monocytes from three peripheral blood samples (PB) of normal donors. To detect point mutations, DNA fragments spanning exon 4 of IDH1 and IDH2 were amplified by nested polymerase chain reaction, and then PCR products were directly sequenced by Sanger sequencing.Results: We studied three splice variants of IDH1: IDH1Va (reported previously), and two variants discovered in the course of this study, IDH1Vb and IDH1Vc. Alignment analyses of these splice variants indicated that the IDH1Vb variant transcripts do not cause a frame shift, and retain native start and stop codons, while IDH1Va and IDH1Vc transcripts have frame shifts that create premature stop codons in the last exon. No splice variants of IDH2 were identified in these patient samples. Of the 242 AML samples (PB-166, BM-76) 51% expressed IDH1Va, 49% expressed IDH1Vb and 23% expressed IDHVc, almost always in combination with full length of IDH1 transcripts (IDH1FL). These IDH1 splice variants were not detected in the normal cells analyzed.We compared expression of IDH1 splice variants, presence of common point mutations, and levels of 2-HG in the 166 patients (Table 1). As expected, patients with point mutations in IDH1 or IDH2 generally had high levels of 2HG in their blood (> average 6104 ng/ml). Seven patients had detectable IDH1/IDH2 point mutations but low 2-HG (< average 222 ng/ml) blood levels. In these patients IDH1Va and IDH1Vb splice variants were detected frequently; Five of 7 patients expressed these variants together and/or in combination with the IDH1-FL mRNA. In contrast, 14 patients had no detectable IDH1/IDH2 mutations but elevated (> average 4430 ng/ml) 2-HG blood levels, twelve of these overexpressed the IDH1Vb splice variant transcripts (Table 1). Six of these 12 patients expressed only IDH1Vb transcripts together with IDH1FL; the average 2HG blood levels in these patients was 2875ng/ml.Conclusion: Though high 2-HG blood levels are correlated with the presence of IDH1/IDH2 mutations, the degree of 2-HG elevation may be altered by other events such as alternative splicing. The expression of IDH1 splice variants was commonly detected in AML samples. Studies to examine the effects of splice variants of IDH1 on 2HG production and transformation are underway. [Display omitted] DisclosuresFathi:Millennium: Research Funding; Seattle Genetics: Advisory Board, Advisory Board Other; Agios: Advisory Board, Advisory Board Other. Yen:Agios: Employment. Kim:Agios: Employment. Zhu:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Yes, same as for being Author of Abstract #70656 Hide Full Conflict-of-Interest Disclosure I have relevant financial relationship(s) to disclose. Yes Name of Organization Type of relationship Agios Pharmaceuticals: Employment. Steensma:Novartis: Consultancy; Celgene: Consultancy; Ariad: Equity Ownership; Amgen: Consultancy. Stone:Agios: Consultancy.

New paradigms in whole genome sequencing: from lab bench to cell phone.

The DNA sequencing & MDx fields have seen dramatic advances since the first draft of the human genome was published, with companies reporting ever faster and cheaper methods. However, despite the race to attain the $1000 genome producing a plethora of exciting technologies, capillary electrophoresis (CE) is still being routinely used for targeted clinical sequencing and expensive real time PCR devices are still the work-horse of the MDx laboratory. QuantuMDx is developing a portable, handheld DNA sequencing device for PGx and infectious disease applications, to provide an alternative to slow and relatively expensive CE DNA sequencing & expensive slow and lab based MDx & CDx.

A multiscale products technique for denoising of DNA capillary electrophoresis signals

Since noise degrades the accuracy and precision of DNA capillary electrophoresis (CE) analysis, signal denoising is thus important to facilitate the postprocessing of CE data. In this paper, a new denoising algorithm based on dyadic wavelet transform using multiscale products is applied for the removal of the noise in the DNA CE signal. The adjacent scale wavelet coefficients are first multiplied to amplify the significant features of the CE signal while diluting noise. Then, noise is suppressed by applying a multiscale threshold to the multiscale products instead of directly to the wavelet coefficients. Finally, the noise-free CE signal is recovered from the thresholded coefficients by using inverse dyadic wavelet transform. We compare the performance of the proposed algorithm with other denoising methods applied to the synthetic CE and real CE signals. Experimental results show that the new scheme achieves better removal of noise while preserving the shape of peaks corresponding to the analytes in the sample.

Epigenotoxicity of environmental pollutants evaluated by a combination of DNA methylation inhibition and capillary electrophoresis–laser-induced fluorescence immunoassay

A variety of environmental pollutants may cause abnormal DNA methylation, which further disturb gene expression. In this work, we developed a rapid and sensitive method for the characterization and identification of the epigenotoxicity of environmental pollutants in terms of DNA methylation. The method combines in vitro inhibition reactions of a model DNA methyltransferase (DNMT) with rapid and sensitive capillary electrophoresis-laser-induced fluorescence (CE-LIF) immunoassays. This method was first assessed using two known DNMT inhibitors, (-)-epigallocatechin-3-gallate and RG108, and then applied to epigenotoxic evaluation of four aldehydes and six benzo-1,4-quinones. It was found that all these electrophilic chemicals could inhibit DNMT activity, probably due to their interactions with the active sites of DNMT. Interestingly, benzo-1,4-quinones displayed more inhibitory effects on DNMT activity than aldehydes. Among the tested six benzo-1,4-quinones, halogenated benzo-1,4-quinone showed higher inhibitory activity than non-halogenated p-benzo-1,4-quinone. Owing to its speed and sensitivity, our method will be potentially applicable for fast epigenotoxic screening of environmental pollutants and mechanistic study of environmental epigenetics.

Does infection play a role in the pathogenesis of granuloma annulare?

Does infection play a role in the pathogenesis of granuloma annulare?

Capillary Electrophoresis of DNA in Hydroxyethylcellulose

Capillary Electrophoresis of DNA in Hydroxyethylcellulose

Integration of rapid DNA hybridization and capillary zone electrophoresis using bidirectional isotachophoresis

We present a method for rapid, sequence-specific detection of multiple DNA fragments by integrating isotachophoresis (ITP) based DNA hybridization and capillary zone electrophoresis (CZE) using bidirectional ITP. Our method leverages the high preconcentration ability of ITP to accelerate slow, second-order DNA hybridization kinetics, and the high resolving power of CZE to separate and identify reaction products. We demonstrate the speed and sensitivity of our assay by detecting 5 pM, 39 nt ssDNA target within 3 min, using a molecular beacon probe. We also demonstrate the feasibility of our assay for multiplexed detection of multiple-length ssDNA targets by simultaneously detecting 39 and 90 nt ssDNA targets.

Integrated microfluidic chip for rapid DNA digestion and time-resolved capillary electrophoresis analysis.

An integrated microfluidic chip is proposed for rapid DNA digestion and time-resolved capillary electrophoresis (CE) analysis. The chip comprises two gel-filled chambers for DNA enrichment and purification, respectively, a T-form micromixer for DNA/restriction enzyme mixing, a serpentine channel for DNA digestion reaction, and a CE channel for on-line capillary electrophoresis analysis. The DNA and restriction enzyme are mixed electroomostically using a pinched-switching DC field. The experimental and numerical results show that a mixing performance of 97% is achieved within a distance of 1 mm from the T-junction when a driving voltage of 90 V/cm and a switching frequency of 4 Hz are applied. Successive mixing digestion and capillary electrophoresis operation clearly present the changes on digesting φx-174 DNA in different CE runs. The time-resolved electropherograms show that the proposed device enables a φx-174 DNA sample comprising 11 fragments to be concentrated and analyzed within 24 min. Overall, the results presented in this study show that the proposed microfluidic chip provides a rapid and effective tool for DNA digestion and CE analysis applications.

Correlation between quantified promoter methylation and enzymatic activity of O 6-methylguanine-DNA methyltransferase in glioblastomas

The DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT, AGT) is a determinant of the resistance of tumor cells to alkylating anticancer agents that target the O(6) position of guanine. MGMT promoter methylation in tumors is regarded as the most common predictor of the responsiveness of glioblastoma to alkylating agents. However, MGMT promoter methylation status has been investigated mainly by methylation-specific PCR, which is a qualitative and subjective assay. In addition, the actual enzymatic activities associated with the methylation status of MGMT have not been explored. In the present study, MGMT promoter methylation in glioblastomas was quantified by bisulfite pyrosequencing, and its correlation with enzymatic activity was determined using a novel quantitative assay for studying the functional activity of MGMT. MGMT enzymatic activity was assessed using fluorometrically labeled oligonucleotide substrates containing MGMT-specific DNA lesions and capillary electrophoresis to detect and quantify these lesions. In comparison with existing traditional assays, this assay was equally sensitive but less time consuming and easier to perform. MGMT promoter methylation was assessed in 41 glioblastomas by bisulfite pyrosequencing, and five samples with different values were chosen for comparison with enzymatic assays. Bisulfite pyrosequencing using primers designed to work in the upstream promoter regions of MGMT demonstrated high quantitative capability and reproducibility in triplicate measurements. In comparative studies, MGMT promoter methylation values obtained by bisulfite pyrosequencing were inversely proportional to the measured enzymatic activity. The present results indicate that the quantification of MGMT methylation by bisulfite pyrosequencing represents its enzymatic activity and thus, its therapeutic responsiveness to alkylating agents.

Effect of Electric Field Modes on the Separation Performance of DNA in Capillary Electrophoresis

Effect of Electric Field Modes on the Separation Performance of DNA in Capillary Electrophoresis

Doing more with less: Implementing direct amplification with the PowerPlex ® 18D System

Time and labor savings continue to be key goals for database and paternity labs. Increasing sample numbers and tightening government budgets necessitate that database laboratories seek process improvements. Similarly, efficiency gains are key for commercial paternity testing organizations to remain competitive. Recently, the STR-typing community has begun to adopt the concept of “direct amplification” from unpurified samples on FTA ® cards. Significant time and labor savings can be achieved by eliminating DNA purification and amplifying samples directly from buccal or blood samples on FTA ® cards. Another time-consuming process is the PCR itself—often three hours or more. The duration of this step often dictates daily workflow for laboratories, and when combined with DNA purification and capillary electrophoresis, the process often requires more than one day. Although thermal cycling is a hands-off step, decreasing sample time on an instrument with rapid amplification allows samples to be processed in a single day. The PowerPlex ® 18D System allows direct amplification and reduced thermal cycling time to provide a simpler and faster genotyping system for database and paternity laboratories. Additionally, the 18 loci amplified represent all markers commonly typed in CODIS laboratories. A validation of the PowerPlex ® 18D System was completed according to SWGDAM guidelines [1] to demonstrate effectiveness and performance of the assay. This testing included sensitivity, concordance, variation of thermal cycling parameters, variation of reaction component concentrations, and tolerance to contaminating substances. The results support that the PowerPlex ® 18D System is a robust and reliable identification assay fit for human identification purposes and the kit has subsequently been approved for use by NDIS CODIS-participating laboratories.

Incorporation of guanosine gels into sieving matrices for length‐ and sequence‐based separation of DNA in capillary electrophoresis

Sieving gels are used in capillary gel electrophoresis to resolve DNA strands of different lengths. For complex samples, however, such as those encountered in metagenomic analysis of microbial communities or biofilms, length-based separation may mask the true genetic diversity of the community since different organisms may contribute same-length DNA with different sequences. There is a need, therefore, for DNA separations based on both the length and sequence. Previous work has demonstrated the ability of guanosine gels (G-gels) to separate four single-stranded DNA 76-mers that differ by only a few A/G base substitutions. The goal of the present work is to determine whether G-gels could be combined with commercial sieving gels in order to simultaneously separate DNA based on both length and sequence. The results are given for the four 76-mers and for a standard dsDNA ladder. Commercial sieving gels were used alone and in combination with G-gels. For the 76-mers, the combined medium was less efficient than the G-gel alone but was able to achieve partial resolution. The combined medium was at least as effective as the sieving gel alone at resolving the denatured DNA ladder and showed indications of sequence-based resolution as well, as supported by MALDI-MS. The results show that the combined sieving gel/G-gel medium retains the selectivity of the individual media, providing a promising approach to simultaneous length- and sequence-based DNA separation for metagenomic analysis of complex systems.