High-throughput genotyping of factor V Leiden mutation by ultrathin-layer agarose gel electrophoresis.

Ultrathin-layer agarose gel electrophoresis is a novel combination of the established methodologies of slab gel electrophoresis and capillary gel electrophoresis. This new format provides a multilane separation platform with rapid analysis time and excellent sensitivity by using laser-induced fluorescence scanning detection system. Sample injection onto the ultrathin-layer separation platform is easily accomplished by membrane mediated loading technology. In this paper, we demonstrate the sensitivity and high-throughput fashion of this novel separation and detection system for rapid genotyping of the coagulation factor V Leiden mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. The PCR amplified fragment from exon 10 of the factor V gene was digested by the Mnl I restriction enzyme, followed by automated ultrathin-layer agarose gel electrophoresis analysis with "in migratio" fluorescent labeling during the separation process. Due to its speed and automation, this method should be considered for large scale screening of factor V Leiden mutation.     

Rapid and sensitive genotyping of dopamine D4 receptor tandem repeats by automated ultrathin-layer gel electrophoresis

Prior studies have revealed possible association between the presence of a seven repeat of the 48 bp variable number tandem repeat polymorphism of the human dopamine D4 receptor gene (DRD4) and some normal and pathological human traits, such as novelty seeking, hyperactivity disorders, and substance abuse. Some reports supported this finding whereas others did not. Incorrect genotyping could be one of the reasons for these controversial results, and might originate from preferential amplification of shorter polymerase chain reaction (PCR) products, resulting in the so-called allele dropout. In this paper we optimized the conditions for simultaneous amplification of shorter and longer amplicons of the 48 bp repeat region of the DRD4 gene in order to avoid the loss of the longer allele and consequent incorrect genotyping, using very low DNA template concentrations and partial replacement of 2'-deoxyguanosine-5'-triphosphate (dGTP) by 2'-deoxyinosine-5'-triphosphate (dITP). The optimized PCR method in combination with high throughput automated ultrathin-layer gel electrophoresis was suitable for rapid genotyping from less than a nanogram DNA using noninvasive sampling (buccal epithelial cells). All detected genotypes are presented, including such rear heterozygotes as the 2 x and 8 x 48 bp repeats in the same sample, showing the reliability of our novel detection method of longer alleles in the presence of shorter alleles.     

ARMS TaqMan real-time PCR for genotyping factor V Leiden mutation in Han Chinese

Factor V Leiden (FV_Leiden) is a missense mutation of 1691 position (G1691A) in exon 10 of FV gene, and being a genetic risk for venous thrombosis. Currently, there are several PCR-based methods for detecting FV_Leiden mutation; however, these methods have disadvantages such as time-consuming, cumbersome steps and potentially hazardous gels. The aims of present study were to develop a simple, time-saving, accurate, and gel-free method, called amplification refractory mutation system (ARMS) TaqMan real-time PCR, for detecting FV_Leiden mutation. We severally designed two specific reverse primers for mutant and wild-type through intentional introduction of mismatched nucleotide at the penultimate 3′ position. Although target amplicon amplification efficiency is reduced, but another corresponding amplicon is almost completely inhibited. Then, specific TaqMan-probe was designed to detect target amplicon. Established method was used to detect 500 unselected samples in Han Chinese, the results showed 499 cases of wild-type and one heterozygote. Afterward, 50 randomly picked wild-type cases and one heterozygote were reexamined by bidirectional DNA sequencing, which is considered as “Gold standard method.” Exhilaratingly, the results detected by the two methods were completely consistent. At last, allelic frequency of FV_Leiden was calculated the in Han Chinese. Given the above results, A FV_Leiden heterozygote has been found in 500 random samples in Han Chinese, and the allelic frequency was 0.1%. In conclusion, the ARMS TaqMan real-time PCR is an ideal detecting system for genotyping FV_Leiden mutation in clinical application, and FV_Leiden mutation exists in Han Chinese despite extremely low prevalence.     

Rapid microwell polymerase chain reaction with subsequent ultrathin-layer gel electrophoresis of DNA

Large-scale genotyping, mapping and expression profiling require affordable, fully automated high-throughput devices enabling rapid, high-performance analysis using minute quantities of reagents. In this paper, we describe a new combination of microwell polymerase chain reaction (PCR) based DNA amplification technique with automated ultrathin-layer gel electrophoresis analysis of the resulting products. This technique decreases the reagent consumption (total reaction volume 0.75-1 microL), the time requirement of the PCR (15-20 min) and subsequent ultrathin-layer gel electrophoresis based fragment analysis (5 min) by automating the current manual procedure and reducing the human intervention using sample loading robots and computerized real time data analysis. Small aliquots (0.2 microL) of the submicroliter size PCR reaction were transferred onto loading membranes and analyzed by ultrathin-layer gel electrophoresis which is a novel, high-performance and automated microseparation technique. This system employs integrated scanning laser-induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. Visualization of the DNA fragments was accomplished by "in migratio" complexation with ethidium bromide during the electrophoresis process also enabling real time imaging and data analysis.     

Multiplex agarose gel electrophoresis system for variable number of tandem repeats genotyping: Analysis example using Mycobacterium tuberculosis

As one genotyping method for Mycobacterium tuberculosis, variable number of tandem repeats (VNTR) is a promising tool to trace the undefined transmission of tuberculosis, but it often requires large equipment such as a genetic analyzer for DNA fragment analysis or CE system to conduct systematic analyses. For convenient genotyping at low cost in laboratories, we designed a multiplex PCR system that is applicable to agarose gel electrophoresis using fluorescent PCR primers. For tuberculosis genotyping by VNTR, the copy quantities of minisatellite DNA must be determined in more than 12 loci. The system can halve laborious electrophoresis processes by presenting an image of two VNTR amplicons on a single lane. No expensive equipment is necessary for this method. Therefore, it is useful even in developing countries.     

Separation of intact pyruvate dehydrogenase complex using blue native agarose gel electrophoresis

We show that the blue native gel polyacrylamide electrophoresis system (BN-PAGE) can be applied to pyruvate dehydrogenase complex (PDC). BN-PAGE has been used extensively to study the multisubunit enzymes of oxidative phosphorylation, as nondenaturing separation in the first dimension maintains holoenzyme integrity. However, the standard protocol was inappropriate for PDC as, at 10 MDa, it is approximately ten times larger than the largest respiratory chain enzyme complex. Therefore, agarose was substituted for polyacrylamide. Moreover, a substantial decrease in salt concentration was necessary to prevent dissociation of PDC. As with standard BN-PAGE, immunoblots of second-dimensional sodium dodecyl sulfate-PAGE (SDS-PAGE) provided more detailed information on specific subunits and subcomplexes. The method was applied to human heart mitochondrial fragments, control cultured human cells, rho0 cells that lack mitochondrial DNA, and two cell lines derived from patients with PDC deficiency. The PDC deficient cell lines showed a clear correlation between amount of PDC holoenzyme and disease severity. In cells lacking mitochondrial DNA, synthesis and assembly of all PDC subunits (all nuclearly encoded) appeared normal, suggesting that respiratory function has no regulatory role in PDC biogenesis. Blue native agarose gel electrophoresis coupled with standard second-dimensional SDS-PAGE provides a new tool to be used in conjunction with biochemical assays and immunoblots of one-dimensional SDS-PAGE to further elucidate the nature of PDC in normal and disease states. Furthermore, other cellular protein complexes of 1 MDa or more can be analysed by this method.     

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.     

Clinical application of a rapid method using agarose gel electrophoresis and Western blotting to evaluate von Willebrand factor protease activity

A method for evaluating the activity of the von Willebrand factor (vWF) protease is described, and a clinical application is illustrated. The procedure utilizes gel electrophoresis, Western blotting, and luminographic detection methods to evaluate the distribution of vWF multimers before and after incubation of clinical samples under conditions that favor proteolysis by this enzyme. Physiologically, the high-molecular-weight multimers of vWF are cleaved by the vWF protease under conditions of high shear stress in parts of the arterial circulation; cleavage of vWF multimers is also observed after exposure of vWF to denaturing agents in vitro and thus can serve as a laboratory test for the activity of the protease. vWF protease activity is decreased or absent in patients with thrombotic thrombocytopenic purpura due to an inhibiting autoantibody, and this leads to high levels of noncleaved vWF and to life-threatening thrombosis, thrombocytopenia and anemia. The assay evaluates the activity of the protease by assessing the cleavage of vWF multimers after patient plasmas are incubated in vitro under denaturing conditions. With the use of these electrophoresis and Western blotting techniques, patient plasmas can be rapidly assessed for the activity of the vWF protease which may aid in the treatment strategy for these patients.     

Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments

DNA fragments up to 9 kb in size were stacked and separated by polyacrylamide gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis, using a discontinuous buffer system. Polyacrylamide gels at pH 8.9, 2 degrees C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 micrograms/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72-23130 bp, while agarose gels at pH 8.5, 25 degrees C, provided well-resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 microgram/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 x TBE (Tris-borate-EDTA) buffer, while that successfully applied to polyacrylamide is seven-fold less than the ionic strength of conventionally used 1 x TBE buffer, with a substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification by Rf, the migration distance relative to a sharply defined "buffer front" (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico-chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid and sensitive silver-lipopolysaccharide staining using PhastSystem in fast horizontal polyacrylamide gel electrophoresis

A rapid and sensitive silver-lipopolysaccharide staining method has been developed by using PhastSystem. The total time of the procedure (including time of Phastgel electrophoresis) is within 2 h. It is at least 10 times faster than the previous reported methods and the sensitivity is also increased.     

Cost-effective interrogation of single nucleotide polymorphisms using the mismatch amplification mutation assay and capillary electrophoresis

The ability to characterize SNPs is an important aspect of many clinical diagnostic, genetic and evolutionary studies. Here, we designed a multiplexed SNP genotyping method to survey a large number of phylogenetically informative SNPs within the genome of the bacterium Bacillus anthracis. This novel method, CE universal tail mismatch amplification mutation assay (CUMA), allows for PCR multiplexing and automatic scoring of SNP genotypes, thus providing a rapid, economical and higher throughput alternative to more expensive SNP genotyping techniques. CUMA delivered accurate B. anthracis SNP genotyping results and, when multiplexed, saved reagent costs by more than 80% compared with TaqMan real-time PCR. When real-time PCR technology and instrumentation is unavailable or the reagents are cost-prohibitive, CUMA is a powerful alternative for SNP genotyping.     

Rapid analysis of covalently and non-covalently fluorophore-labeled proteins using ultra-thin-layer sodium dodecylsulfate gel electrophoresis

Gel electrophoresis is one of the most frequently used tools for the separation of complex biopolymer mixtures. In recent years, there has been considerable activity in the separation and characterization of protein molecules by sodium dodecylsulfate (SDS) gel electrophoresis with particular interest in using this technique to separate on the basis of size and to estimate molecular mass and protein purity. Although the method is informative, it is cumbersome, time consuming and lacks automation. In this paper we report an automated, high-performance SDS gel electrophoresis system that is based on electric-field-mediated separation of SDS-protein complexes using an ultra-thin-layer platform. The integrated fiber optic bundle-based scanning laser-induced fluorescence detection technology readily provided high sensitivity, real-time detection of the migrating solute molecules. Rapid separations of covalently and non-covalently labeled proteins were demonstrated in the molecular mass range 14,000 to 205,000 in less than 9 and 16 min, respectively. Excellent quantitation and lane-to-lane migration time reproducibility were found for all the solute components using the multilane separation platform. The limit of detection was found to be 1.5-3 ng/band for both labeling methods, with excellent linearity over a six times serial double-dilution range. Molecular mass calibration plots were compared for both covalently and non-covalently labeled proteins. A linear relationship was found between the molecular mass and electrophoretic mobility in the case of covalently labeled samples, while a non-linear relationship was revealed for the non-covalently labeled samples.     

Rapid high-resolution electrophoresis of multimeric von Willebrand Factor using a thermopiloted gel apparatus

Rapid and highly reproducible nonreducing agarose gel electrophoresis (NRAGE) of von Willebrand Factor (vWF) multimers was performed using a thermostated minigel apparatus that monitors and precisely controls internal gel temperature. The substitution of lithium dodecyl sulfate (LiDS) for sodium dodecyl sulfate (SDS) allowed electrophoresis to be performed below the 16 degrees C Krafft point of SDS and facilitated NRAGE of vWF over the entire range of 0-35 degrees C. Internal gel temperature was regulated by a thermocouple probe inserted directly into the gel during electrophoresis which interfaced with a thermopilot that continually measures and adjusts temperature to within +/- 0.5 degrees C. At 10 degrees C operative temperature, NRAGE at 1.5% agarose concentration was completed in 20 min at 250 V. Electrophoresis could be performed in only 10 min at 500 V, but at such high voltages, localized temperature fluctuations as much as 6 degrees C resulted in perturbation of banding patterns in those vicinities. In the optimized method, both high molecular weight multimers and proteolytic fragments of vWF were separable suggesting clinical applicability of this system for the diagnosis of von Willebrand Disease and thrombotic thrombocytopenic purpura.     

Separation and purification of puerarin using beta-cyclodextrin-coupled agarose gel media

The isoflavonoid puerarin, a well-known traditional Chinese drug, has been purified in one step from an extract of Radix puerariae (root of the plant Pueralria lobata) by adsorption chromatography on an epichlorohydrin polymerized beta-cyclodextrin ligand coupled to brominated allyl-group substituted Sepharose HP. Acetic acid (10%) was used as the mobile phase and the optimum loading capacity was around 1.2 mg crude extract/ml packed gel. The purity of the collected puerarin was about 98% with a recovery of about 62%.     

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2× or 4×48 base pair repeats) of the dopamine D₄ receptor gene (D₄DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.     

Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip

We report on the development of a hybrid polydimethylsiloxane (PDMS)-glass microchip for genetic analysis by functional integration of polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE), and on related temperature control systems for PCR on a PDMS-glass hybrid microchip. The microchip was produced by molding PDMS against a microfabricated master with comparatively simple and inexpensive methods. PCR was successfully carried out on the PDMS-glass hybrid microchip with 500 bp target of lambdaDNA and the amplified gene was subsequently analyzed by CGE on the same PDMS-glass microchip. The chip could be considered as an inexpensive single-use apparatus compared to glass or silicon-made microchips for the same purpose.     

Rapid analysis of atorvastatin calcium using capillary electrophoresis and microchip electrophoresis

In this work, a capillary electrophoretic method for the rapid quantitation of atorvastatin (AT) in a lipitor tablet was investigated and developed. Method development included studies of the effect of applied potential, buffer concentration, buffer pH, and hydrodynamic injection time on the electrophoretic separation. The method was validated with regard to linearity, precision, specificity, LOD, and LOQ. The optimum electrophoretic separation conditions were 25 mM sodium acetate buffer at pH 6, with a separation voltage of 25 kV using a 50 microm capillary of 33 cm total length. Sodium diclofenac was used as an internal standard. Analysis of AT in a commercial lipitor tablet by electrophoresis gave quite high efficiency, coupled with an analysis time of less than 1.2 min in comparison to LC. Once the separation was optimized on capillary, it was further miniaturized to a microchip platform, with linear imaging UV detection using microchip electrophoresis (MCE). Linear imaging UV detection allowed for real-time monitoring of the analyte movement on chip, so that the optimum separation time could be easily determined. This microchip electrophoretic method was compared to the CE method with regard to speed, efficiency, precision, and LOD. This work represents the most rapid and first reported analysis of AT using MCE.     

Stoichiometry-dependent formation of quantum dot-antibody bioconjugates: a complementary atomic force microscopy and agarose gel electrophoresis study

The unique advantages of quantum dot (QD) bioconjugates have motivated their application in biological assays. However, physical characterization of bioconjugated QDs after surface modification has often been overlooked. Here, biotinylated antibodies (biotin-IgG) were attached to commercial streptavidin-conjugated quantum dots (strep-QDs) at different stoichiometric ratios, and these QD bioconjugates were characterized with atomic force microscopy and discontinuous sodium dodecyl sulfate agarose gel electrophoresis (SDS-AGE). The results from these complementary analytical techniques showed that the molar ratio determined the relative sizes, molecular weights and morphologies of the QD bioconjugates. Additionally, the novel discontinuous SDS-AGE analysis confirmed specific binding between biotin-IgG and strep-QDs. Researchers who design QD bioconjugates for cell-based assays should consider stoichiometry-dependent differences in the physical properties of their QD bioconjugates.     

Simultaneous Ferguson plot analysis, using electrophoresis on a single agarose pore gradient gel, of DNA fragments contained in a mixture.

A recent study has demonstrated the feasibility of obtaining Ferguson plots in agarose gel electrophoresis, using a single pore gradient gel. We now report three remedies for defects in the previous experimental approach: (i) UV-absorbing media for density stabilization of the gel is avoided by replacing 5-(N-2,3-dihydroxypropylacetamido)-2,4,6-triiodo-N,N'-bis(2,3-dihy droxypropyl) isophthalamide (Nycodenz) with heavy water; this renders the method applicable to ethidium bromide-labeled DNA. (ii) The density stabilizing medium is kept from having an effect on field strength. (iii) Data collection by uninterrupted time-lapse photography is possible by using an apparatus with a quartz window. These three measures make the method practical for the gel electrophoretic identification and physical characterization of DNA species, potentially up to 50 kb in size.