Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions

Separations of phiX-174/HaeIII DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5-5 microg/mL), (ii) voltage programming (125-375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris-borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8,000,000) under isocratic conditions have been completely resolved by either stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 mM TB buffers, pH 10.0, high-speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/ Bg/l digest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.     

A new strategy for optimizing sensitivity, speed, and resolution in capillary electrophoretic separation of DNA

DNA separations were performed in poly(ethylene oxide) (PEO) solutions prepared in 100 mM Tris-boric acid (TB) buffers using a capillary filled with TB buffers with concentrations up to 2.5 M, pH 10.0. The electroosmotic flow (EOF) increased with increasing the concentration of TB buffers till 1.5 M as a result of decreasing PEO adsorption on the capillary wall. At high TB concentrations (> 1.5 M), the peaks corresponding to small DNA fragments (11 and 8 base pairs) became sharper and were detected. Relative standard deviations of the EOF coefficient and the migration times of the DNA fragments were all less than 1% using a capillary filled with TB buffers at concentrations higher than 1.5 M. When separations were performed at different pH values of PEO solutions and TB buffers, better results in terms of sensitivity, speed, and resolution were generally achieved. The fluorescence intensity of the 2176 bp fragment obtained at pH values of TB buffers/PEO solutions 10.0/8.2 was 27-fold of that at pH values 8.2/8.2. The enhancement was related to effects of pH and borate on fluorescence intensity, DNA conformation, stacking, and interactions with the capillary wall. Using a capillary filled with 400 mM TB buffers, pH 10.0, the separation of DNA (pBR 322/HaeIII digest, pBR 328/Bg/I digest and pBR 328/HinfI digest) in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 9.0, at 375 V/cm was accomplished in less than 18 min.     

Maximization of injection volumes for DNA analysis in capillary electrophoresis

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.     

Modification of poly(methyl methacrylate) microchannels for highly efficient and reproducible electrophoretic separations of double-stranded DNA

This paper deals with dynamic coating of the microchannels fabricated on poly(methyl methacrylate) (PMMA) chips and DNA separation by microchip electrophoresis (MCE). After testing a number of polymers, including 2-hydroxyethyl cellulose, hydroxypropylmethyl cellulose, different sizes of poly(ethylene oxide) (PEO), and poly(vinyl pyrrolidone) (PVP), we found that coating of the PMMA microchannels with PEO(Mr = 6.0 x 10(5) g/mol) on the first layer is essential to minimize the interaction of DNA with PMMA surface. To achieve high efficiency, multilayer coating of PMMA chips with PEO, PVP, and PEO containing gold nanoparticles [PEO(GNP)] is important. A 2-(PEO-PVP)-PEO(GNP) PMMA chip, which was repeatedly coated with 1.0% PEO and 5.0% PVP twice, and then coated with 0.75% PEO(GNP) each for 30 min, provided a high efficiency (up to 1.7 x 10(6) plates/m) for the separation of DNA markers V (pBR 322/HaeIII digest) and VI (pBR 328/BgiI digest and pBR 328/HinfI digest) when using 0.75% PEO(GNP). With such a high efficiency, we demonstrated the separation of hsp65 gene fragments of Mycobacterium HaeIII digests by MCE within 90 s. The advantages of this approach to DNA analysis include ease of filling the microchannel with 0.75% PEO(GNP), rapidity, and reproducibility.     

The impact of a plug of salts on the analysis of large volumes of dsDNA by capillary electrophoresis

A partially filling technique for the analysis of DNA markers and polymerase chain reaction (PCR) products by capillary electrophoresis in the presence of electroosmotic flow using polymer solutions is presented. Either after or prior to the sample injection, a plug of salts at high pH was hydrodynamically injected. During the separation, poly(ethylene oxide) (PEO) solution entered the capillary. We have found that the position, length, and composition of the plugs affect the sensitivity, resolution, and speed on the analysis of PhiX-174/HaeIII DNA restriction fragments or a DNA mixture (pBR 322/HaeIII digest, pBR 328/BglI digest and pBR 328/HinfI digest) with different degrees. Through careful evaluation of the impact of anions and cations on the analysis of DNA, we have suggested that the optimal condition is applying a plug consisting of 32 mM NaCl and 0.01 M NaOH at 30 cm height for 60 s after sample injection. In the presence of such a plug, PEO adsorption reduces, and thus the separation is faster, as well as the sensitivity improves. Using this condition, the analysis of a DNA mixture (injected at 30 cm for 360 s) containing ten different PCR products amplified after 17 cycles was complete in 25 min. About a 2000-fold improvement in the sensitivity was achieved when compared to that by a conventional method (10 s injection) without applying a plug.     

Separation of double-stranded DNA fragments by capillary electrophoresis: Impacts of poly(ethylene oxide), gold nanoparticles, ethidium bromide, and pH

The separation of DNA by capillary electrophoresis using poly(ethylene oxide) (PEO) containing gold nanoparticles (GNPs) is presented. The impacts of PEO, GNPs, ethidium bromide (EtBr), and pH on the separation of double-stranded DNA have been carefully explored. Using a capillary dynamically coated with 5.0% poly(vinylpyrrolidone) and filled with 0.2% PEO containing 0.3 x GNPs (the viscosity less than 15 cP), we have demonstrated the separation of DNA markers V and VI within 5 min at pH 8.0 and 9.0. In terms of resolution and reproducibility, GNPs have a greater impact on the separation of DNA at pH 9.0. Resolution improvements for large DNA fragments (> 300 base pairs, bp) are greater than those for small ones in the presence of GNPs. It is important to point out that reproducibility is excellent (relative standard deviations for the migration times less than 0.5%) and thus no further dynamic coating is required in at least 20 consecutive runs in the presence of GNPs. Using 0.2% PEO (pH 9.0) containing 0.3 x GNPs, the separation of DNA fragments ranging in size from 21 to 23,130 bp was accomplished in 7 min. The results presented in this study show the advantage of PEO containing GNPs for DNA separation, including rapidity, high resolving power, excellent reproducibility, and ease of filling capillaries.     

Analysis of large-volume DNA markers and polymerase chain reaction products by capillary electrophoresis in the presence of electroosmotic flow

We have demonstrated on-line concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large-volumes DNA samples, PEO solutions entered a capillary filled with 400 mM Tris-borate (TB) buffers by EOF and acted as sieving matrices. DNA fragments stacked between the sample zone and PEO solutions. Because sample matrixes affected PEO adsorption on the capillary wall, leading to changes in EOF, migration time, concentration, and resolving power varied with the injection length. When injecting phiX174 RF DNA-HaeIII digest prepared in 5 mM Tris-HCl buffer, pH 7.0, at 250 V/cm, peak height increased linearly as a function of injection volume up to 0.9 microl (injection time 150 s). The sensitivity improvement was 100-fold compare to that injected at 25 V/cm for 10 s (0.006 microl). When injecting 1.54 microl of GeneScan 1000 ROX, the sensitivity improvement was 265-fold. The sensitivity improvement was 40-fold when injecting 0.17 microl DNA sample containing pBR 322/HaeIII, pBR 328/BglI, and pBR 328/HinfI digests prepared in phosphate-buffered saline. This method allows the analysis of polymerase chain reaction (PCR) products amplified after 17 cycles when injecting 0.32 microl (at 30 cm height for 300 s). The total analysis time was shorter (91.6 min) than that (119.6 min) obtained from injecting PCR products after 32 cycles for 10 s.     

On-column concentration and separation of double-stranded DNA by gradient capillary electrophoresis

We describe the separation of dsDNA by capillary electrophoresis in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO). Using 1.0% PEO, the separation of DNA fragments with sizes ranging from 51 bp to 23 kbp has been achieved in less than 12 min, which is better than conventional methods (in the absence of EOF) in terms of speed and resolution. In order to concentrate and separate the DNA sample, gradient changes in the concentrations of PEO and ethidium bromide (EtBr) have been conducted. Different concentrations of PEO solutions are injected to the polyethylene tubes by pressure, where they enter the capillary by EOF. Because the large DNA fragments migrate faster towards the cathode end under counterflow conditions, the introduction sequence is from low to high concentrations of PEO solutions after sample injection. Using the gradient CE approach, the separations of the DNA sample injected at 30 cm height for times up to 120 s have been demonstrated. The linearity between injection time and peak height shows that the DNA fragments stacked during migration from the sample zone to PEO. We found that stacking efficiency is greater when the analysis was performed by simultaneously changing the PEO and EtBr concentration, compared to individual changes in PEO concentration.     

Stepwise capillary electrophoretic separation of DNA fragments using poly(ethylene oxide) solutions in the presence of electroosmotic flow.

Single-base resolution in the separation of DNA markers V and VI was achieved in the presence of electroosmotic flow (EOF), using poly(ethylene oxide) (PEO) solutions containing ethidium bromide (EtB) under isocratic conditions. Furthermore, a new approach called stepwise capillary electrophoresis (SCE) has been developed for DNA analysis, including stepwise changes in PEO concentration, EtB concentration as well as both PEO and EtB concentrations, wherein the EOF was used to introduce different PEO solutions into the capillary during the separation. DNA fragments smaller than 80 bp were both detected under isocratic conditions using 20 micrograms/ml EtB, and SCE using 1 and 20 micrograms/ml EtB, but not under isocratic conditions using 1 microgram/ml EtB. Resolution and speed of the DNA separation in SCE were different from those obtained from isocratic means, indicating that DNA underwent different concentrations of PEO and EtB in SCE. For example, DNA fragments with 458 and 504 base pairs (bp) were partially resolved in SCE, but not under isocratic conditions. The results further suggest that it is worth developing gradient techniques for widening the separation range and enhancing resolution in DNA analysis.     

On-column preconcentration and separation of DNA fragments using polymer solutions in the presence of electroosmotic flow

We demonstrated DNA preconcentration and separation in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large volumes of DNA samples into a capillary filled with free tris(hydroxymethyl)aminomethane (Tris)-borate (TB) buffers, PEO solutions entered the capillary by EOF and acted as sieving matrices. In contrast to conventional methods (in the absence of EOF), controlling the EOF was also useful for resolution optimization. We have found that PEO adsorption on the capillary wall was more pronounced when low ionic strength buffers were used. Thus, the EOF decreased with increasing injection length, which led to longer migration times and changes in resolution and stacking efficiency. All resolution values were higher than 1.5 when 1.0 microg/mL DNA samples were injected at 240 V/cm for 60 s (0.67 microL). In addition, as low as 0.015 microg/mL DNA samples (an about 66-fold increase in sensitivity) were detected when the injection was performed at 250 V/cm for 60 s.     

Fast and sensitive diagnosis of thalassemia by capillary electrophoresis

This paper demonstrates the diagnosis of -thalassemia by capillary electrophoresis in conjunction with laser-induced fluorescence using poly(ethylene oxide) (PEO) solutions in the presence of electroosmotic flow (EOF). During the electrophoretic separation, PEO solution entered a capillary from the anodic vial by EOF. The separation of a mixture of the polymerase chain reaction (PCR) products (330 and 334 base pairs) from a healthy person and a -thalassemia patient was accomplished within 15 min at 15 kV using 1.5% PEO containing 2 M urea at 30 °C. The electropherogram patterns instead of migration times were used to diagnose -thalassemia, with an accuracy of 100% for the analyses of 11 blood samples from suspected patients. After injecting a large volume of the mixture to the capillary filled with 800 mM Tris-borate buffer (pH 10.0), the DNA fragments stacked due to increases in viscosity and sieving when migrating into 1.5% PEO solution. As a result of improved sensitivity, only 15 PCR cycles were required when using 500 ng of DNA templates. The results shown in this study indicate the potential of this simple, rapid, and cost-effective method for the diagnosis of -thalassemia.Abbreviations CE Capillary electrophoresis - EOF Electroosmotic flow - EtBr Ethidium bromide - LIF Laser-induced fluorescence - PCR Polymerase chain reaction - PEO Poly(ethylene oxide) - TRIS Tris(hydroxymethyl)aminomethane - TB TRIS-borate     

Improved separation of double-stranded DNA fragments by capillary electrophoresis using poly(ethylene oxide) solution containing colloids

The analysis of double-stranded (ds) DNA fragments by capillary electrophoresis (CE) using poly(ethylene oxide) (PEO) solution containing gold nanoparticles (GNPs) is presented, focusing on evaluating size dependence of the GNPs and PEO on resolution and speed. To prevent the interaction of the capillary wall with DNA, the capillary was dynamically coated with polyvinylpyrrolidone. Using different PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm, we have achieved reproducible, rapid, and high-resolution DNA separations. The results indicate that the sizes of PEO and GNPs as well as the concentration of PEO affect resolution. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO (8 MDa) containing 56 nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp using 0.05% PEO (2 MDa) containing 13 nm GNPs or 0.05% PEO (4 MDa) containing 32 nm GNPs. With very low viscosity (< 15 cP), automatic replacement of the sieving matrices is easy, indicating a great potential for high-throughput DNA analysis using capillary array electrophoresis systems.     

聚环氧乙烷无胶筛分毛细管电泳分离宽分子量范围DNA片段

在无胶筛分毛细管电泳中,以聚环氧乙烷为筛分介质,用硅烷化处理的毛细管柱（31.2 cm×75 μm有效长度21.0 cm）分离DL5000 DNA Marker（DNA长度为100~5000 bp）,研究筛分介质浓度、缓冲液pH、分离电压和溴化乙锭浓度对分离双链DNA片段的影响,优化出分离100~5000 bp DNA片段的最佳条件。毛细管电泳的最佳条件为PEO浓度0.5%、缓冲液pH值8.0、电压12 kV、溴化乙锭浓度3.0 μg/mL。此条件下,对山梨醇脱氢酶基因（SDH）和乙烯受体基因（ETR1）的聚合酶链式反应（PCR）扩增产物同时检测,分离、鉴定效果良好。     

Analysis of double-stranded DNA by microchip capillary electrophoresis using polymer solutions containing gold nanoparticles

The impact of gold nanoparticles (GNPs) on the microchip electrophoretic separation of double-stranded (ds) DNA using poly(ethylene oxide) (PEO) is described. Coating of the 75-microm separation channel on a poly(methyl methacrylate) (PMMA) plate in sequence with poly(vinyl pyrrolidone), PEO, and 13-nm GNPs is effective to improve reproducibility and resolution. In this study, we have also found that adding 13-nm GNPs to 1.5% PEO is extremely important to achieve high resolution and reproducibility for DNA separation. In terms of the stability of the GNPs, 100 mM glycine-citrate buffer at pH 9.2 is a good buffer system for preparing 1.5% PEO. The separation of DNA markers V and VI ranging in size from 8 to 2176 base pairs has been demonstrated using the three-layer-coated PMMA microdevice filled with 1.5% PEO containing the GNPs. Using these conditions, the analysis of the polymerase chain reaction products of UGT1A7 was complete in 7 min, with the relative standard deviation values of the peak heights and migration times less than 2.3% and 2.0%, respectively. In conjunction with stepwise changes of the concentrations of ethidium bromide (0.5 and 5 microg/ml), this method allows improved resolution and sensitivity for DNA markers V and VI.     

Separation of amino acids and amines by capillary electrophoresis using poly(ethylene oxide) solution containing cetyltrimethylammonium bromide

We describe simultaneous analysis of naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and amine derivatives by capillary electrophoresis in conjunction with light-emitting diode-induced fluorescence (LEDIF) detection using poly(ethylene oxide) (PEO) containing cetyltrimethylammonium bromide (CTAB). In the presence of CTAB and acetonitrile (ACN), adsorption of PEO on the capillary wall is suppressed, leading to generation of a fast and reproducible electroosmotic flow (EOF). In order to optimize separation resolution and speed, 100 mM Tris–borate solution (pH 7.0) containing 20 mM CTAB and 25% ACN was used to fill the capillary and to prepare 1.2% PEO that entered the capillary via EOF. The analysis of 14 NDA-amino acid and -amine derivatives by this approach is rapid (< 4 min), efficient ((0.9–6.4) × 10⁵ theoretical plates), and sensitive (the LODs (S/N = 3) range from 9.5 to 50.5 nM). The RSD values (n = 5) of the migration times and peak heights of the analytes for the intraday analysis are less than 1.5 and 1.2%, respectively. We have validated the practicality of this approach by quantitative determination of 10 amino acids and amines in a beer samples within 4 min.     

Screening of mixed poly(ethylene oxide) solutions for microchip separation of double-stranded DNA using an orthogonal design approach

We describe a quick and systematic optimization of molecular weights (MWs) and concentrations of a series of mixed poly(ethylene oxide) (PEO) matrices for separating specific double-stranded DNA fragments on polymethylmethacrylate-based microchips by using an orthogonal design (ORD) approach. The mixed matrices are composed of PEOs in four MW ranges (M(w) 8 x 10(6), 1 x 10(6), 4 x 10(5), and 1 x 10(5)) with varying concentration ratios. In the mixed solutions, PEO with an intermediate MW of 4 x 10(5) is found to be a dominant factor for separating small DNA fragment pairs (e.g., 82 and 88 bp), while PEO with a high MW of 8 x 10(6) plays an important role in separating intermediate and large fragments (e.g., 271 and 281 bp, 506 and 517 bp, 7 and 10 kbp). High-concentration PEO mixtures give better resolution for short fragments, while dilute PEO mixtures show better resolution for long fragments. The optimized matrices are suitable for high-resolution separation of multiplex polymerase chain reaction-amplified products and restriction digest fragments ranging in size from 20 bp to 40 kbp within 4 min at a constant field strength of 177 V/cm. The experimental results indicate the robustness and speediness of the ORD to screen the contribution of PEO MWs and to tune optimally the PEO concentration ratio of different MWs with reference to the performance of specific DNA fragments separated.     

Clay-enhanced DNA separation in low-molecular-weight poly(N,N-dimethylacrylamide) solution by capillary electrophoresis

The effect of the separation medium in capillary electrophoresis consisting of a low-molecular-mass poly(N,N-dimethylacrylamide) (PDMA) solution on the DNA separation by adding a small amount of montmorillonite clay into the polymer matrix is presented. On the separation of the pBR322/HaeIII digest, both the resolution and the efficiency were increased by adding 2.5-5.0 x 10(-5) g/mL clay into the 5% w/v PDMA with a molecular mass of only 100 K. Moreover, there was no increase in the migration time of DNA fragments. Similar results were observed by using a C-terminated pGEM-3Zf(+) sequencing DNA sample in a sequencing buffer. Experimental data also showed that the addition of clay increased the viscosity of the polymer solution. We attribute this effect to the structural change of the polymer matrix caused by the exfoliated clay sheets, whereby the thin clay sheets function like a "dynamic cross-linking plate" for the PDMA chains and effectively increase the apparent molecular mass of PDMA.     

Stacking and separation of protein derivatives of naphthalene-2,3-dicarboxaldehyde by CE with light-emitting diode induced fluorescence detection

We describe the stacking and separation of proteins by CE under discontinuous conditions in conjunction with light-emitting diode induced fluorescence (LEDIF) detection using a violet LED at 405 nm. The proteins were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to form NDA-protein derivatives prior to CE-LEDIF analysis. During the separation, poly(ethylene oxide) (PEO) solution containing CTAB enters from the cathodic inlet to the capillary via electroosomotic flow (EOF). The optimum conditions are: the capillary was filled with 50 mM glycine buffer (pH 9.0) containing 1.0 mM CTAB, NDA-protein derivatives were prepared in deionized water containing 1.0 mM CTAB, and 0.6% PEO was prepared in 50 mM glycine (pH 9.0) containing 2.0 mM CTAB. The analysis of four NDA-protein derivatives is fast (<3 min), with RSD <1.5% in terms of migration time. In order to improve the sensitivity of NDA-protein derivatives, a stacking approach based on increases in viscosity and electric field, as well as sieving was applied. The efficient stacking approach provides LODs (S/N = 3) of 2.41, 0.59, 0.61, and 4.22 nM for trypsin inhibitor, HSA, beta-lactoglobulin, and lysozyme, respectively. In addition, we also applied the stacking approach to determination of the concentration of HSA in one urine sample, which was determined to be 0.31 +/- 0.05 microM (n = 3).     

EFFECT OF OVERLAP CONCENTRATION AND PERSISTENCE LENGTH ON DNA SEPARATION IN POLYMER SOLUTIONS BY ELECTROPHORESIS

The persistence length and the overlap concentration(c~*) of poly(ethylene oxide)(PEO) and hydroxyethylcellulose(HEC) with similar molecular weight in 1×TBE buffer were studied by laser light scattering and viscometry.Their effect on DNA separation was investigated by capillary electrophoresis.It was determined that the persistence length of HEC was at least 5 times higher than that of PEO.Therefore,the c~* of HEC was smaller than that of PEO by a factor of ca.2.5.It was also found that the c~* values de...     

A novel spectrofluorimetric method for the determination of DNA

A new simple, selective and sensitive fluorescence quenching method was developed to determine nucleic acids (DNA) with the 9-anthracenecarboxylic acid (ACA)-cetyl trimethyl-ammonium bromide (CTAB) system. The fluorescence intensity of ACA was decreased by the addition (CTAB). However, the fluorescence intensity of the system increased dramatically when DNA was added to the solution. The fluorescence enhancement is probably based on the DNA interaction with CTAB. Under the optimum conditions, the changes of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.08-1.0 microg mL(-1) for CT (calf thymus) DNA or FS (fish sperm) DNA. Its detection limits are 0.02 microg mL(-1) for CT DNA and 0.019 microg mL(-1) for FS DNA. Based on this approach, a new quantitative method for DNA assay is presented in this paper.