Telomerase activity measurement in magnetically captured epithelial cells: comparison of slab-gel and capillary electrophoresis

We have compared telomerase activity measurements by slab-gel and capillary electrophoresis in cultured cells (A549 and H125 human cancer cell lines) and in cells isolated from clinical peripheral blood specimens epithelial cells of patients with lung and esophageal cancer. Telomerase activity was determined using the telomerase repeat amplification protocol (TRAP) assay with phosphoimager scanning of slab-gels and by laser-induced fluorescence capillary electrophoresis (LIF-CE). Experiments using A549 and H125 cells were performed to determine the reproducibility of each method and to identify the contribution of each stage of the TRAP/polymerase chain reaction (PCR) assay to the variability. In these experiments, it was found that more than half of the overall variability (coefficient of variation, CV = 35%) of the slab-gel method and almost all of the overall variability (CV = 20%) of the CE method was due to the PCR stage of the TRAP assay. In the clinical samples, classification as positive or negative was by visual inspection of the slab-gel and CE electropherograms for the presence of the characteristic 6 base-pair TRAP ladder and by GeneScan analysis of the CE. We examined several criteria including the use of 3, 4, or 5 TRAP bands as the definition of a positive test. Using the slab-gel method, the 5-band criterion gave 40% sensitivity with 100% specificity (no false positives in inactive controls). The CE method yielded a comparable 38% sensitivity and 100% specificity using this criterion. These data indicate that detection of telomerase activity in epithelial cells isolated from peripheral blood has a useful level of sensitivity and specificity and may be useful in the detection and monitoring of aerodigestive cancers. However, analysis by slab-gel is cumbersome and the precision is poor (inter-replicate CV = 20%) compared to LIF-CE (CV = 5%). A high-throughput CE-LIF detection platform will be indispensable for validation studies of telomerase activity measurements.     

Detection of telomerase activity by combination of telomeric repeat amplification protocol and electrochemiluminescence assay

A highly sensitive telomerase detection method that combines telomeric repeat amplification protocol （TRAP） and magnetic beads based electrochemiluminescence （ECL） assay has been developed. Briefly, telomerase recognizes biotinylated telomerase synthesis primer （B-TS） and synthesizes extension products, which then serve as the templates for PCR amplification using B-TS as the forward primer and tris-（2′2′-bipyridyl） ruthenium （TBR） labeled ACX （TBR-ACX） as the reversed primer. The amplified product is captured on streptavidin-coated paramagnetic beads and detected by ECL. Telomerase positive HeLa cells were used to validate the feasibility of the method. The experimental results showed down to 10 cancer cells can be detected easily. The method is a useful tool for telomerase activity analysis due to its sensitivity, rapidity, safety, high throughput, and low cost. It can be used for screening a large amount of clinical samples.     

Influence of fluorophor dye labels on the migration behavior of polymerase chain reaction--amplified short tandem repeats during denaturing capillary electrophoresis.

The determination of the length of polymerase chain reaction (PCR)-amplified short tandem repeats (STRs) by denaturing capillary electrophoresis (CE) is a standard procedure for purposes of genotyping. We show that dye-specific mobility anomalies exist for 5'-fluorophor-labelled single-stranded DNA (ssDNA) fragments in CE using the performance-optimized polymer 4 (POP4) buffer sieving matrix, containing the entangled poly(N,N-dimethylacrylamide) polymer, urea, and 2-pyrrolidinone. The dye-specific retardation effects relative to coseparated GeneScan-500 [TAMRA] standard fragments can lead to wrong genotyping, even for allele-specific fragments of pentanucleotide STRs, when comparing the relative calculated sizes of identical fragments, labelled with rhodamine (ROX, TAMRA) or fluorescein dyes (FAM, 6-FAM, HEX, JOE, NED, TET): The size of fluorescein dye-labelled fragments of appr. 100 b in length appears to be smaller by up to 6.5 b. This effect becomes more dramatic with decreasing size: a 6-FAM-labelled 24-mer oligonucleotide appeared to be smaller by 11 b. In contrast, in classical urea/polyacrylamide slab-gel electrophoresis only a small dye-specific retardation of identical fragments is observed. The dye-specific effects are superimposed by weaker size and sequence-dependent anomalies of fragment mobility. Therefore, in denaturing CE the coseparation of a defined allele ladder labelled with the same dye as the unknown sample fragments remains the method of choice for accurate genotyping.     

Detection of telomerase activity in high concentration of cell lysates using primer-modified gold nanoparticles

Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its use has been significantly limited when performed directly in complex, interferant-laced samples. In this work, we report a modification of the TRAP assay that allows the detection of high-fidelity amplification of telomerase products directly from concentrated cell lysates. Briefly, we covalently attached 12 nm gold nanoparticles (AuNPs) to the telomere strand (TS) primer, which is used as a substrate for telomerase elongation. These TS-modified AuNPs significantly reduce polymerase chain reaction (PCR) artifacts (such as primer dimers) and improve the yield of amplified telomerase products relative to the traditional TRAP assay when amplification is performed in concentrated cell lysates. Specifically, because the TS-modified AuNPs eliminate most of the primer-dimer artifacts normally visible at the same position as the shortest amplified telomerase PCR product apparent on agarose gels, the AuNP-modified TRAP assay exhibits excellent sensitivity. Consequently, we observed a 10-fold increase in sensitivity for cancer cells diluted 1000-fold with somatic cells. It thus appears that the use of AuNP-modified primers significantly improves the sensitivity and specificity of the traditional TRAP assay and may be an effective method by which PCR can be performed directly in concentrated cell lysates.     

Multiplex polymerase chain reaction analysis of Glu-1 high-molecular-mass glutenin genes from wheat by capillary electrophoresis with laser-induced fluorescence detection

The unique bread-making properties of wheat are closely correlated with composition and quantity of high-molecular-mass (HMW) glutenin subunits encoded by the Glu-1 genes. We report the development of a multiplex polymerase chain reaction (PCR) method to identify bread wheat genotypes carrying HMW glutenin allele composition of Glu-1 complex loci (Glu-A1, Glu-B1 and Glu-D1) by capillary electrophoresis(CE) with laser-induced fluorescence (LIF) detection. Two triplex primer sets of HMW glutenin subunit genes were examined. An automated and rapid CE-LIF technique is helpful in the multiplex PCR optimization process. Two fluorescent intercalating dyes (EnhanCE, and YO-PRO-1) are compared for detection of DNA fragments. Amplified DNA fragments of HMW glutenin Glu-1 genes were well separated both by agarose slab-gel electrophoresis and CE, and revealed minor differences between the sequences of 1Ax2*, 1Axnull, 1Bx6, 1Bx7, 1Bx17 and 1Dx5 genes. Moreover, CE technique requires samples of smaller volumes in comparison to slab-gel electrophoresis, and data can be obtained in less than 20 min. There was a very high concordance in the assessment of the molecular size of PCR-generated DNA markers. Fast and accurate identification of molecular markers of Glu-1 genes by CE-LIF can be an efficient alternative to standard procedure separation for early selection of useful wheat genotypes with good bread-making quality.     

Nonradioactive telomerase activity assay by microchip electrophoresis: privileges to the classical gel electrophoresis assay

The present study accents on the privileges of microchip-based electrophoresis to the conventional gel electrophoresis in separation of telomerase repeat amplification protocol/polymerase chain reaction (PCR) ladder products obtained in telomerase-catalyzed reaction in cancer cells. We try to clarify the interpretation of the results obtained by both electrophoretic procedures and to avoid misinterpretation as a result of PCR-dependent artefacts.     

Real-time PCR detection of telomerase activity using specific molecular beacon probes

Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures, reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly. Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nanomaterials and chip-based nanostructures for capillary electrophoretic separations of DNA

Capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) using polymer solutions are two of the most powerful techniques for the analysis of DNA. Problems, such as the difficulty of filling polymer solution to small separation channels, recovering DNA, and narrow separation size ranges, have put a pressure on developing new techniques for DNA analysis. In this review, we deal with DNA separation using chip-based nanostructures and nanomaterials in CE and MCE. On the basis of the dependence of the mobility of DNA molecules on the size and shape of nanostructures, several unique chip-based devices have been developed for the separation of DNA, particularly for long DNA molecules. Unlike conventional CE and MCE methods, sieving matrices are not required when using nanostructures. Filling extremely low-viscosity nanomaterials in the presence and absence of polymer solutions to small separation channels is an alternative for the separations of DNA from several base pairs (bp) to tens kbp. The advantages and shortages of the use of nanostructured devices and nanomaterials for DNA separation are carefully addressed with respect to speed, resolution, reproducibility, costs, and operation.     

Comparative analysis of tea catechins and theaflavins by high-performance liquid chromatography and capillary electrophoresis

This paper describes the simultaneous determination of catechins and theaflavins in green and black teas, using reversed-phase high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The tea polyphenols analyzed included (+)-catechin, catechin gallate, (-)-epicatechin, epicatechin-3-gallate, epigallocatechin, epigallocatechin-3-gallate, theaflavin, theaflavin-3-monogallate, theaflavin-3'-monogallate and theaflavin-3,3'-gallate. These polyphenols together with six other tea ingredients such as caffeine, adenine, theophylline, quercetin, gallic acid and caffeic acid were separated within 27 min by HPLC and in less than 10 min by CE. The optimal analytical conditions of both chromatographic methods were investigated for the convenience and reliability for routine analysis. Both HPLC and CE were found to be reliable and compatible. The reproducibility of the within-day assay using both methods was generally >90%. The day-to-day variation of retention time was <5% for HPLC, while the variation of migration time for CE was <2%. The analysis time of CE was three-times faster, however it is five-times less sensitive than HPLC, which has detection limits of 0.05 microg/ml and 0.5 microg/ml for catechins and theaflavins, respectively.     

Separation of single-stranded DNAs using DNA conjugates having different migration properties in capillary electrophoresis

A probe-regulated simultaneous separation (PRESS) using capillary electrophoresis (CE) was developed for separating single-stranded (ss) DNAs. We synthesized two DNA conjugate probes, -(5'-TGTGTGTGT-3')p-AAm(q)- and -(5'-GCCACCAGC-3')m-AAm(n)-, by copolymerizing 5'-methacryloyl-modified ssDNA with acrylamide (AAm), and characterized them in detail. The two probes showed lower electrophoretic mobilities than 5'-methacryloyl-modified ssDNAs. Furthermore, -(5'-TGTGTGTGT-3')p-AAm(q)- showed slightly faster electrophoretic mobility toward the anode than -(5'-GCCACCAGC-3')m-AAm(n)- due to its higher molar fraction of negatively-charged ssDNA. We successfully separated target ssDNAs having the same chain length by using two ssDNA conjugate probes that showed different electrophoretic mobilities, although the separation of these ssDNAs was difficult in conventional capillary electrophoresis systems.     

Cover Picture: Electrophoresis 14'2011

Issue no. 14 is a regular issue consisting of 17 contributions distributed over 3 distinct parts. Part I has 7 articles describing studies on proteins and proteomics including measuring protein mobility using laser Doppler electrophoresis, high sensitivity protein analysis by FESI‐CE‐MALDI‐MS, protocol for SDS‐PAGE separation of myosin heavy chain isoforms, proteomics analysis of a spring wheat cultivar in response to prolonged cold stress, analysis of changes in the 20S proteasome, treatment of acute lymphoblastic leukemia with L‐asparaginase, and improved method for immunostaining of mucin. Part II is on nucleic acids and has 4 contributions on multiplex PCR‐CE analysis, copy number variation, ethnic difference, SNPs, CE‐SSCP, allelic ladder and SNaPshot technique. Part III has 6 articles on various fundamentals and methodologies, including nanofluidics, nanopore sensing, sequential injection setup for CIEF combined with MS detection, determination of imatinib mesylate in chronic myeloid leukemia patients by CE, UV detection of plasma malondialdehyde using CE‐FASI, enantioseparation of basic drugs by CE using clarithromycin lactobionate chiral selector and CE determination of bioactive constituents in Herba Houttuyniae. Featured articles include: Advances in the measurement of protein mobility using laser Doppler electrophoresis – the diffusion barrier technique (( 10.1002/elps.201100108 )) Improved method for immunostaining of mucin separated by supported molecular matrix electrophoresis by optimizing the matrix composition and fixation procedure (( 10.1002/elps.201000608 )) Strategy for high‐fidelity multiplex DNA copy number assay system using capillary electrophoresis devices (( 10.1002/elps.201100093 )) Electrokinetic particle translocation through a nanopore containing a floating electrode (( 10.1002/elps.201100050 )) Sequential injection setup for capillary isoelectric focusing combined with MS detection (( 10.1002/elps.201100012 ))     

Capillary electropherograms for restriction fragment length polymorphism of Helicobacter pylori

Rapid identification of Helicobacter pylori strains is of importance for diagnosis and then treatment of duodenal and gastric ulcers. We developed a CE approach for the analysis of RFLP of the PCR products of urease (UreAB) gene and flagellin A (FlaA) gene fragments. Prior to CE analysis, the 2.4-kbp UreAB and 1.5-kbp FlaA PCR products were digested with the restriction enzymes HaeIII and HhaI, respectively. The DNA fragments were then separated by CE in conjunction with laser-induced fluorescence detection using poly(ethylene oxide) in the presence of electroosmotic flow. The DNA fragments range in sizes 259-1831 bp and 12-827 bp for UreAB and FlaA restriction fragments, respectively. Of 27 samples, the CE approach provided five and ten different RFLP patterns of the HaeIII and HhaI digests. The RFLP of PCR products of the two genes allow great sensitivity of identification of H. pylori strains. When compared with slab gel electrophoresis, the present CE approach provides advantages of rapidity (within 6 min per run), simplicity, and automation. The preliminary results have shown great practicality of the CE approach for screening H. pylori strains.     

Capillary electrophoresis combining three-step multiplex polymerase chain reactions for diagnosing α-thalassemia

Capillary electrophoresis (CE) is the most useful tool for DNA separation because of its high resolution. In this study, different kinds of polymers were used to evaluate the separation efficiency by analyzing a 200-bp DNA ladder. Under optimized CE conditions, the CE separation was performed by DB-17 capillary. The running buffer was a 1× TBE buffer containing 0.6% w/v poly(ethylene oxide) (PEO) (Mw: 8,000,000) and 1?μM YO-PRO-1; applied voltage was -10?kV (detector at anode side) and the separation temperature was 25°C. Under these optimal conditions, 15 DNA fragments with sizes ranging from 0.2 to 3.0?kb were resolved within 11.5?min and the RSD of migration time were less than 0.55% (n=3). This method, combined with three-step multiplex PCR, was applied to detect five α-thalassemia deletions, including -α(3.7) , -α(4.2) , - -(SEA) , - -(FIL) and - -(THAI) . A total of 21 patients diagnosed with α-thalassemia were analyzed using this developed method and all results agreed with those already obtained by gel electrophoresis.     

Studies on characterization, telomerase inhibitory properties and G-quadruplex binding of η6-arene ruthenium complexes with 1,10-phenanthroline-derived ligands

Two arene ruthenium complexes [Ru(η(6)-C(6)H(6))(p-MOPIP)Cl](+)1 and [Ru(η(6)-C(6)H(6))(p-CFPIP)Cl](+)2, where p-MOPIP = 2-(4-methoxyphenyl)-imidazo[4,5f][1,10] phenanthroline and p-CFPIP = 2-(4-trifluoromethylphenyl)-imidazo[4,5f][1,10] phenanthroline, were prepared and the interactions of these compounds with DNA oligomers 5'-G3(T2AG3)3-3'(HTG21) have been studied by UV-vis and circular dichroism (CD) spectroscopy, gel mobility shift assay, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay and telomeric repeat amplification protocol (TRAP) assay. The results show that both complexes can induce the stabilization of quadruplex DNA but complex 1 is a better G-quadruplex binder than complex 2. The two ruthenium complexes tested led to an inhibition of the enzyme telomerase and complex 1 was the significantly better inhibitor. A novel visual method has been developed for making a distinction between G-quadruplex DNA and double DNA by our Ru complexes binding hemin to form the hemin-G-quadruplex DNAzyme. Furthermore, in vitro cytotoxicity studies showed complex 1 exhibited quite potent antitumor activities and the greatest inhibitory selectivity against cancer cell lines.     

Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.     

Synthesis and evaluation of analogues of 10H-indolo[3,2-b]quinoline as G-quadruplex stabilising ligands and potential inhibitors of the enzyme telomerase

We report here the synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of several rationally-designed quindoline analogues, substituted at the 2- and 7- positions. The ability of these compounds to interact with and stabilise an intramolecular G-quadruplex DNA against increases in temperature was evaluated by a fluorescence-based (FRET) melting assay. The resulting T(m) values were found to correlate with their potency for telomerase inhibition, as measured in an in vitro telomerase TRAP assay. The interactions of a number of compounds with a quadruplex DNA molecular structure were simulated by molecular modelling methods. It is concluded that this class of compound represents a new chemical type suitable for further development as telomerase inhibitors.     

Electrophoresis of pharmaceutical proteins: status quo

The biotechnology industry has undergone rapid growth in recent years largely due to the development and success of protein-based therapeutics for a wide range of disorders. Similar to traditional pharmaceuticals, characterization of a therapeutic protein for its physicochemical properties, process monitoring and lot release is crucial. Electrophoresis in the slab-gel format has and continues to be a mainstay of the protein laboratory; and more recently, CE has begun to make significant inroads for protein analysis in industrial settings. This review focuses on the electrophoresis of proteins with an emphasis on protein-based therapeutics in the capillary, slab-gel and to a lesser extent, the microchip format. Reported applications of electrophoresis at several stages of the biopharmaceutical industry covering the period of 2000-2005 will be discussed.     

Facile assay of telomerase activity utilizing a DNA-detectable chemiluminogenic reagent.

Telomerase shows increased activity in most human cancers and germ line cells, but not in normal human somatic cells. We describe a novel chemiluminescence method for the facile assay of telomerase activity in human cells. The telomerase substrate was incubated with the cell lysate containing various amounts of telomerase, and then the telomerase product was amplified by the polymerase-chained reaction (PCR). The PCR products were separated from the excess substrate, primer and deoxyribonucleotide triphosphates by a centrifugal filter, which distinguished different molecular sizes. The isolated products were reacted with a DNA-detectable chemiluminogenic reagent, 3,4,5-trimethoxyphenylglyoxal. The proposed assay method gave linearity for the telomerase activity in 100 to 10000 cells (r2=0.997), and allowed the assay not only of lower activity, but also of higher activity of telomerase without the requirement of any special labeled-PCR primers in the assay system.     

Development and validation of a capillary electrophoresis method for the characterization of protegrin IB-367.

A capillary electrophoresis (CE) method was developed to characterize protegrin IB-367, an antimicrobial peptide being developed for the treatment of oral mucositis and for other topical applications. The electrophoretic purity and levels of potential impurities/degradation products of IB-367 drug substance are determined by CE using area normalization. Electrophoresis parameters were optimized to allow optimal resolution, reproducibility and minimal analysis time. The separation and resolution between this polycationic peptide and truncated analogs determined by the CE method was much greater than those by the HPLC methods. In addition, the CE methods separates the potential impurities/degradation products from each other while the HPLC methods failed to resolve them. The CE method was validated in the aspects of accuracy, precision, linearity, range, limit of detection, limit of quantitation, specificity, system suitability and robustness. An internal standard was used for the quantitation purpose. The selection criteria of the internal standard as well as the method validation results are presented. The truncated peptide analogs were used to demonstrate the specificity of the method. These analogs were also used to evaluate the limit of quantitation of potential impurities. The relative response factors of these analogs were assessed to determine area normalization feasibility. System suitability tests were established.     

Detection of apolipoprotein E genotypes by capillary electrophoresis

The apolipoprotein E (apo-E) genotype of an individual is of significant relevance in the associated risk of developing cardiovascular disease and late-onset Alzheimer's disease. Detection of the six common apo-E genotypes is based on the restriction fragment length polymorphisms (RFLPs) arising from the abolition or creation of HhaI restriction sites within an amplified target DNA sequence of the apo-E gene. Genomic DNA was extracted from leukocytes, a 230 bp target sequence within the apo-E gene was amplified by polymerase chain reaction (PCR) and digested with HhaI, and the restricted DNA fragments separated by capillary electrophoresis (CE). This was performed on the BioFocustrade mark 3000 automated CE system equipped with an experimental laser-induced fluorescence (LIF) detector (Bio-Rad Laboratories, Hercules, CA), using capillaries (27 cm length, 75 mum i.d.) coated internally with polyaminoacryloylethoxyethanol. The analysis buffer (2xTris borate-EDTA, pH 8.3) was supplemented with a proprietary sieving polymer and 0.05 muM thiazole orange six. Samples were injected electrophoretically. Separations were carried out at 40 degrees C under constant voltage, and the emitted fluorescence detected at 515 nm. Restriction fragment lengths of the cleaved PCR products were estimated from the migration times, with a 20/100 bp ladder (Bio-Rad Laboratories 20/100 bp molecular ruler) serving as reference. Six different reproducible patterns were obtained for the six common apo-E genotypes, with good resolution of the component restriction fragments. The calculated sizes of the separated peaks closely corresponded with the predicted restricted fragment lengths for each specific genotype. We believe this is the first published report demonstrating the feasibility of automating the post-PCR detection of the apo-E RFLPs(2). This methodology overcomes the most labour-intensive step in apo-E genotyping, thus making it amenable to routine clinical application.