| 1. |
- Bus, Magdalena M., et al.
(författare)
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Multiplex pyrosequencing of InDel markers for forensic DNA analysis
- 2016
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 37:23-24, s. 3039-3045
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Tidskriftsartikel (refereegranskat)abstract
- The capillary electrophoresis (CE) technology is commonly used for fragment length separation of markers in forensic DNA analysis. In this study, pyrosequencing technology was used as an alternative and rapid tool for the analysis of biallelic InDel (insertion/deletion) markers for individual identification. The DNA typing is based on a subset of the InDel markers that are included in the Investigator (R) DIPplex Kit, which are sequenced in a multiplex pyrosequencing analysis. To facilitate the analysis of degraded DNA, the polymerase chain reaction (PCR) fragments were kept short in the primer design. Samples from individuals of Swedish origin were genotyped using the pyrosequencing strategy and analysis of the Investigator (R) DIPplex markers with CE. A comparison between the pyrosequencing and CE data revealed concordant results demonstrating a robust and correct genotyping by pyrosequencing. Using optimal marker combination and a directed dispensation strategy, five markers could be multiplexed and analyzed simultaneously. In this proof-of-principle study, we demonstrate that multiplex InDel pyrosequencing analysis is possible. However, further studies on degraded samples, lower DNA quantities, and mixtures will be required to fully optimize InDel analysis by pyrosequencing for forensic applications. Overall, although CE analysis is implemented in most forensic laboratories, multiplex InDel pyrosequencing offers a cost-effective alternative for some applications.
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| 2. |
- Drabek, Jiri, et al.
(författare)
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Design and validation of an STR hexaplex assay for DNA profiling of grapevine cultivars
- 2016
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 37:23-24, s. 3059-3067
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Tidskriftsartikel (refereegranskat)abstract
- Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser-induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high-throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.
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| 3. |
- El Deeb, Sami, et al.
(författare)
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Recent advances in capillary electrophoretic migration techniques for pharmaceutical analysis (2013-2015)
- 2016
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 37:12, s. 1591-1608
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Forskningsöversikt (refereegranskat)abstract
- This review updates and follows-up a previous review by highlighting recent advancements regarding capillary electromigration methodologies and applications in pharmaceutical analysis. General approaches such as quality by design as well as sample injection methods and detection sensitivity are discussed. The separation and analysis of drug-related substances, chiral CE, and chiral CE-MS in addition to the determination of physicochemical constants are addressed. The advantages of applying affinity capillary electrophoresis in studying receptor-ligand interactions are highlighted. Finally, current aspects related to the analysis of biopharmaceuticals are reviewed. The present review covers the literature between January 2013 and December 2015.
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| 4. |
- Krais, Annette, et al.
(författare)
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Determination of genomic N3-methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser-induced fluorescence.
- 2019
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 40:11, s. 1535-1539
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Tidskriftsartikel (refereegranskat)abstract
- Methylating substances alter DNA by forming N3-methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), we synthesized the N3mT-3'-phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1,000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N'-nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5-hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5-methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE-LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT
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| 5. |
- Krais, Annette, et al.
(författare)
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Sensitive detection of hydroxymethylcytosine levels in normal and neoplastic cells and tissues.
- 2019
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 40:9, s. 1293-1297
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Tidskriftsartikel (refereegranskat)abstract
- A new sensitive analytical method using capillary electrophoresis with laser induced fluorescence (CE-LIF) was applied for the simultaneous detection of DNA methylation and hydroxymethylation levels in human cancers of different origin. DNA hydroxymethylation, measured as 5-hydroxymethylcytosine (5hmC) levels, was decreased in gliomas with mutation in the isocitrate dehydrogenase 1 (IDH1) gene when compared to IDH1-wildtype gliomas. Independent from IDH1 mutation, 5hmC levels were decreased in lung carcinomas when compared to normal lung tissue. Reduced DNA hydroxymethylation was also observed upon dedifferentiation in cultured murine embryonic fibroblasts. Our data show that reduced DNA hydroxymethylation is related to cellular dedifferentiation and can be detected in various types of cancers, independent from the IDH1 mutation status. Quantitative determination of altered 5hmC levels may therefore have potential as a biomarker linked to cellular differentiation and tumorigenesis.
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| 6. |
- Lee, Jung Soo, et al.
(författare)
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Stiffness measurement of nanosized liposomes using solid-state nanopore sensor with automated recapturing platform
- 2019
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 40:9, s. 1337-1344
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Tidskriftsartikel (refereegranskat)abstract
- This paper describes a method to gauge the stiffness of nanosized liposomes – a nanoscale vesicle – using a custom-made recapture platform coupled to a solid-state nanopore sensor. The recapture platform electrically profiles a given liposome vesicle multiple times through automated reversal of the voltage polarity immediately following a translocation instance to re-translocate the same analyte through the nanopore – provides better statistical insight at the molecular level by analyzing the same particle multiple times compared to conventional nanopore platforms. The capture frequency depends on the applied voltage with lower voltages (i.e., 100 mV) permitting higher recapture instances than at higher voltages (>200 mV) since the probability of particles exiting the nanopore capture radius increases with voltage. The shape deformation was inferred by comparing the normalized relative current blockade ((Formula presented.) at the two voltage polarities to that of a rigid particle, i.e., polystyrene beads. We found that liposomes deform to adopt a prolate shape at higher voltages. This platform can be further applied to investigate the stiffness of other types of soft matters, e.g., virus, exosomes, endosomes, and accelerate the potential studies in pharmaceutics for increasing the drug packing and unpacking mechanism by controlling the stiffness of the drug vesicles.
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| 7. |
- Mikkonen, Saara, et al.
(författare)
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Computer simulations of sample preconcentration in carrier-free systems and isoelectric focusing in microchannels using simple ampholytes
- 2015
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Ingår i: Electrophoresis. - : Wiley-VCH Verlagsgesellschaft. - 0173-0835 .- 1522-2683. ; 36:19, s. 2386-2395
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Tidskriftsartikel (refereegranskat)abstract
- In this work, electrophoretic preconcentration of protein and peptide samples in microchannels was studied theoretically using the 1D dynamic simulator GENTRANS, and experimentally combined with MS. In all configurations studied, the sample was uniformly distributed throughout the channel before power application, and driving electrodes were used as microchannel ends. In the first part, previously obtained experimental results from carrier-free systems are compared to simulation results, and the effects of atmospheric carbon dioxide and impurities in the sample solution are examined. Simulation provided insight into the dynamics of the transport of all components under the applied electric field and revealed the formation of a pure water zone in the channel center. In the second part, the use of an IEF procedure with simple well defined amphoteric carrier components, i.e. amino acids, for concentration and fractionation of peptides was investigated. By performing simulations a qualitative description of the analyte behavior in this system was obtained. Neurotensin and [Glu1]-Fibrinopeptide B were separated by IEF in microchannels featuring a liquid lid for simple sample handling and placement of the driving electrodes. Component distributions in the channel were detected using MALDI- and nano-ESI-MS and data were in agreement with those obtained by simulation. Dynamic simulations are demonstrated to represent an effective tool to investigate the electrophoretic behavior of all components in the microchannel.
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| 8. |
- Mikkonen, Saara, et al.
(författare)
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Inverse cationic ITP for separation of methadone enantiomers with sulfated beta-cyclodextrin as chiral selector
- 2019
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Ingår i: Electrophoresis. - : WILEY. - 0173-0835 .- 1522-2683. ; 40:5, s. 659-667
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Tidskriftsartikel (refereegranskat)abstract
- Chiral ITP of the weak base methadone using inverse cationic configurations with H+ as leading component and multiple isomer sulfated beta-CD (S-beta-CD) as leading electrolyte (LE) additive, has been studied utilizing dynamic computer simulation, a calculation model based on steady-state values of the ITP zones, and capillary ITP. By varying the amount of acidic S-beta-CD in the LE composed of 3-morpholino-2-hydroxypropanesulfonic acid and the chiral selector, and employing glycylglycine as terminating electrolyte (TE), inverse cationic ITP provides systems in which either both enantiomers, only the enantiomer with weaker complexation, or none of the two enantiomers form cationic ITP zones. For the configuration studied, the data reveal that only S-methadone migrates isotachophoretically when the S-beta-CD concentration in the LE is between about 0.484 and 1.113 mM. Under these conditions, R-methadone migrates zone electrophoretically in the TE. An S-beta-CD concentration between about 0.070 and 0.484 mM results in both S- and R-methadone forming ITP zones. With >1.113 mM and < about 0.050 mM of S-beta-CD in the LE both enantiomers are migrating within the TE and LE, respectively. Chiral inverse cationic ITP with acidic S-beta-CD in the LE is demonstrated to permit selective ITP trapping and concentration of the less interacting enantiomer of a weak base.
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| 9. |
- Nejdl, Lukas, et al.
(författare)
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Label-free bead-based metallothionein electrochemical immunosensor
- 2015
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 36:16, s. 1894-1904
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Tidskriftsartikel (refereegranskat)abstract
- A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 L/min). In stationary arrangement it is possible to detect MT concentrations up to 30ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 g/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.
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| 10. |
- Paul, Prasanta, et al.
(författare)
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A simple, low-cost and robust capillary zone electrophoresis method with capacitively coupled contactless conductivity detection for the routine determination of four selected penicillins in money-constrained laboratories.
- 2018
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 39:20, s. 2521-2529
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Tidskriftsartikel (refereegranskat)abstract
- A simple and robust capillary zone electrophoresis method was developed and validated for the determination of amoxicillin and clavulanate, ampicillin, phenoxymethyl penicillin (Pen V) as well as flucloxacillin. Capacitively coupled contactless conductivity detection was employed as detection mode that makes CE a simple and economic tool for money-constrained laboratories. The developed method is straightforward and user-friendly. It offers good sensitivity and sufficient selectivity for the routine assay of the selected penicillins. The repeatabilities were <1.9% RSD for relative peak areas and <1% RSD for migration times for all the analytes. The method showed good linearity (R2 > 0.995) within the 80-120% range of the target concentration (0.5 mg/mL) for each antibiotic. The accuracy of the method, evaluated by standard fortification at three levels, was good for all the analytes. An extended robustness study was performed by varying ±10% of the optimum value of TRIS concentration, l-histidine concentration and temperature in a full factorial design at two levels. This was to evaluate larger than usual variability of factors on the assay value, in order to better cover potential global variance in lab conditions and equipment. Finally, the method was applied for the determination of percent (%) content of all antibiotics in available formulations.
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