Biomedical subjects

W L Tseng

Publications and source records attributed to W L Tseng.

9 recordsLinked to original sources

PubMed2001

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.

Base Sequence↗

PubMed2001

Regulation of electroosmotic flow and electrophoretic mobility of proteins for concentration without desalting.

Proteins were concentrated and separated in 0.6% poly(ethylene oxide) (PEO) solution using a capillary filled with Tris-borate (TB) buffer prior to analysis and detected by laser-induced native fluorescence using a pulsed Nd:YAG laser. During the concentration and separation, PEO solution entered the capillary by electroosmotic flow. When proteins dissolved in high salts (phosphate-buffered saline) were separated using 0.6% PEO solution prepared in 200 mM TB buffer, pH 9.0, the limits of detection (LODs) at signal-to noise ratios=3 for carbonic anhydrase (CA) and alpha-lactalbumin (alpha-lac) were on the levels of sub microM and microM, respectively. The LOD values compared to those obtained in 38 mM TB buffer were relatively high, which is likely due to salt quenching, Joule heating and poor stacking. To improve sensitivity for analysis of proteins in high-conductivity media, two on-line concentration approaches without desalting were developed. When using a capillary filled with 1.5 M TB buffer, pH 10.0, and PEO solution prepared in 800 mM TB buffer, pH 9.0, the LOD values for CA and alpha-lac were 13.8 nM and 126.0 nM, respectively, which were about 4.7 and 11.2-fold sensitivity enhancements compared to those obtained by a conventional hydrodynamic injection (30 cm height for 10 s), respectively. The sensitivity was further improved by injecting a short plug of low pH buffer after protein injection using a capillary filled with 1.5 M TB buffer, pH 10.0, and PEO solution prepared in 400 mM TB buffer, pH 9.0. A linear relationship between the peak height and the injection volume up to 0.81 microl was obtained and the LOD values for CA and alpha-lac were down to 4.7 and 37.8 nM.

Buffers↗

PubMed2001

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.

Boric Acids↗

PubMed2001

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.

Bacteriophage phi X 174↗

PubMed2000

On-line concentration and separation of proteins by capillary electrophoresis using polymer solutions.

Proteins were separated in 0.6% poly(ethylene oxide) (PEO) solutions using a capillary filled with buffers prior to analysis and were detected by laser-induced native fluorescence using a pulsed Nd:YAG laser. PEO solutions entered the capillary by electroosmotic flow (EOF) during the separation. The composition and concentration of the buffer affected the adsorption of PEO molecules on the capillary surface and, consequently caused changes in the EOF. Short separation times (< 7 min) were achieved on a sample solution of five proteins in a 0.6% PEO solution containing 5 microg/mL ethidium bromide using a capillary pre-filled with 100 mM TRIS-borate (TB) buffers (pH 10,0). We also extended this method for on-line concentration and separation of proteins. Proteins dissolved in low-conductivity media stacked in both TB buffers and in PEO solutions. The peak height was proportional to the injection volume up to 2.1 microL using an 80-cm capillary filled with 400 mM TB buffers. Using large injection volumes (2.1 microL), we achieved a limit of detection (S/N = 3) of 31 pM for carbonic anhydrase, which was a 1696-fold sensitivity enhancement compared to a conventional injection method (1 kV for 10 s). In high-conductivity media (urine matrix), stacking occurred at the boundary between the sample zone and PEO solutions. A urine sample without any pretreatment was analyzed, and after stacking, several peaks were detected. Spiking the urine sample with human serum albumin (HSA) affected the fluorescent intensity of some analytes as a result of interaction with HSA.

Electrophoresis, Capillary↗

PubMed2000

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow.

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.

Buffers↗

PubMed2000

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.

Animals↗

PubMed1999

The epidemiology of Japanese encephalitis on Taiwan during 1966-1997.

Japanese encephalitis (JE) is an endemic disease in Taiwan. A mass vaccination program of children against JE was first implemented in 1968. Along with general improvements in various aspects of living conditions over the years, the program has brought JE well under control. The main characteristics of JE epidemiology in Taiwan in the past 3 decades are as follows. The transmission mode remains unchanged-that is, the amplification stage of the virus in pigs is followed by a human epidemic each year. The frequency of JE incidence has dropped significantly. The incidence rate of confirmed cases was 2.05 per 100,000 in 1967, the highest in record, and merely 0.03 per 100,000 in 1997. Confirmed cases occur sporadically all over the island. The peak of the epidemic season has shifted from August in the 1960s to June since the 1980s. The age distribution of confirmed cases has shifted gradually from mainly children to adults. Vaccine efficacy for those having received more than 2 doses of the vaccine is estimated to be about 85%.

Adult↗

PubMed1997

[Detection of neutralizing antibodies to Japanese encephalitis virus by enzyme-linked immunosorbent assay (ELISA)].

Competitive ELISA was used for the detection of neutralizing antibody to JE. Based on the principle that human serum JE antibody competed with JE monoclonal antibody (MAb) for JE antigen, it was found that 3 JE MAbs (E3-3, NPF-5 and NNN-5) were suitable for competitive ELISA for the detection of JE neutralizing antibody. The sensitivity of cometitive ELISA for 29 JE confirmed serum specimens with titer of plaque reduction neutralization test (PRNT) was checked to be 82.1% (23/28). The specificity of E3-3 MAb to JE used in competitive ELISA was 100%. Correlation coefficient of JE confirmed cases of 57 hemagglutination inhibition (HI) titers in 1995 and 37 PRNT titers in 1994 compared with competitive ELISA were 0.744 and 0.732, respectively. Compared the competitive ELISA titers of 154 sera of healthy people with PRNT titers, the results showed that 70% of the sera could be detected by competitive ELISA which saved a lot of time and manpower.

Antibodies, Viral↗