Po-Ling Chang

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.

Capillary Electrophoretic Restriction Fragment Length Polymorphism Patterns for the Mycobacterial hsp65 Gene

ABSTRACT PCR-restriction fragment length polymorphism (RFLP) analysis is a nonprobe method for the rapid identification of Mycobacterium species. We demonstrate the separation of DNA or restriction fragments digested from the mycobacterial gene encoding the 65-kDa heat shock protein (hsp65) by capillary electrophoresis (CE). By using a pair of unlabeled primers, Tb11 and Tb12, and only one restriction enzyme, HaeIII, we investigated a total of 52 reference and clinical strains encompassing 12 Mycobacterium species. The electrophoretic separation of high-resolution CE required <20 min and was capable of identifying fragments as small as 12 bp. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE, and the real sizes were deduced from the sequence analysis. Distinct differentiations were also well demonstrated between some species and subspecies by an extra HaeIII digestion site. With the advantage of the complete RFLP pattern available from CE, it appears to be more convenient to use an electropherogram rather than performing the cumbersome slab gel electrophoresis plus diagnostic algorithm to identify Mycobacterium species. Beyond the agarose and polyacrylamide gel electrophoresis, high-resolution CE provides an alternative for rapid identification of Mycobacterium species that is feasible for automation and routine use without the need for costly probes.

Comparative microRNA detection from precursor-microRNA-transfected hepatocellular carcinoma cells by capillary electrophoresis with dual-color laser-induced fluorescence.

A dual‐LIF (dLIF) setup combined with CE for microRNA (miRNA) detection is proposed in this study. An argon ion laser (488 nm) and a solid state laser (640 nm) were chosen to excite the fluorescent dye‐labeled DNA probe after splinted ligation of miRNA. The crosstalk of emission spectrum of Alex Fluor 488 and Alex Fluor 647 is minimized with a zero crosstalk matrix for Alex Fluor 647 to 488 channels. The linear ranges of the device for the fluorescent dye‐labeled DNA probe were both from 1.0 nM to 0.1 pM. The limits of detection for Alexa Fluor 488‐labeled DNA and Alex Fluor 647‐labeled DNA were 9.3 and 31 fM, respectively. The detection of specific miRNA has been accomplished by combining splinted ligation with the fluorescent dye‐labeled oligonucleotides. The linear range for the synthetic miRNA is from 1.0 nM to 1.0 pM. Without PCR amplification, CE‐dLIF was applied to discriminate a pre‐miR‐10b*‐transfected cells (contains precursor miR‐10b*) from hepatocellular carcinoma cell (control cells). Therefore, this result indicates CE‐dLIF has great potential to provide a rapid comparative assay for miRNAs detection.

Quantitation of branched‐chain amino acids in ascites by capillary electrophoresis with light‐emitting diode‐induced fluorescence detection

Branched‐chain amino acids (BCAAs) are one of the important biomarkers for monitoring liver disease such as hepatitis or hepatoma. In this communication, we present the determination of the concentrations of BCAA in ascites by CE light‐emitted diode‐induced fluorescence (LEDIF) using 1.5% m/v poly(ethylene oxide) (average Mv: ∼8 000 000 g/mol) that was prepared in 10 mM sodium tetraborate solution (pH 9.3). Naphthalene‐2,3‐dicarboxaldehyde was used to derivatize 15 amino acids (AAs) to form naphthalene‐2,3‐dicarboxaldehyde (NDA)‐AA derivatives prior to CE analysis. The separation of 15 NDA‐AA derivatives was accomplished within 15 min, with RSD values of <5.8% (within‐day) and 7.4% (between‐days) with respect to their migration times. The limits of detection for the tested BCAAs ranged from 10.6 to 10.9 nM. We determined the concentrations of three BCAAs – leucine, isoleucine and valine – in ascites by applying a standard addition method, with recovery percentages ranging from 93.9 to 111%. The results obtained from this CE‐LEDIF method is in good agreement with those by a gold standard method using an AA analyzer. We have found that the concentrations of the three BCAAs in ascites obtained from patients suffering from liver diseases were lower than those from healthy individuals. Our approach is highly efficient, sensitive, and cost‐effective, which holds great potential for the diagnosis of liver diseases.

Rapid screening of the heterogeneity of DNA methylation by single-strand conformation polymorphism and CE-LIF in the presence of electro-osmotic flow.

DNA methylation is a complex event in epigenetic studies because of both the large CpG islands present upstream of the promoter region and the different distribution of DNA methylation despite similar methylation levels. For this reason, we proposed a fast, cost‐effective method for the screening of DNA methylation based on SSCP and CE‐LIF. In this study, the PCR products that were amplified from bisulfite‐treated genomic DNA were denatured at 94°C, followed by immediate chilling in ice water to form the ssDNA. The ssDNA were separated by 1.5% poly(ethylene oxide) (Mavg 8 000 000 Da) in the presence of EOF according to the different conformations represented by their unique methylation states. This result demonstrated that four hepatocellular carcinoma cell lines represented a different heterogeneity of DNA methylation and could be distinguished by SSCP‐CE. The results obtained from SSCP‐CE also corresponded with those obtained from combined bisulfide restriction analysis and methylation‐sensitive high‐resolution melting analysis. Therefore, the proposed SSCP‐CE method may potentially be used for rapid screening for determination of the heterogeneity of DNA methylation in further epigenetic studies and clinical diagnosis.

Strategies of fluorescence staining for trace total ribonucleic acid analysis by capillary electrophoresis with argon ion laser‐induced fluorescence

In this work, five fluorescent dyes (SYTO‐9, SYBR Green I, SYBR Green II, SYBR Safe, and SYBR Gold) were used as both on‐column and precolumn stains for total RNA analysis by CE‐LIF with Ar ion laser excitation. In the on‐column RNA stain, the SYTO‐9 provided the highest fluorescence intensity and the lowest detectable concentration, as low as 10 pg/μL, while the SYBR Green II and SYBR Gold were adsorbed on the poly(ethylene oxide) thus affected the separation efficiency. As a precolumn stain, SYBR Gold was the most sensitive among the five dyes due to the strong affinity between the dye and RNA molecules. As a result, a single‐cell quantity of RNA (10‒30 pg per cell) could be detected by CE‐LIF with precolumn staining by SYBR Gold. Because of the great savings of fluorescent dye using precolumn stain (one button dye may use for one million stain), this method is the best strategy for RNA staining in terms of cost‐effectiveness and sensitivity.

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 ΦX‐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.