Rapid sizing of DNA and analysis of single cells using capillary electrophoresis.

摘要

In chapter 1, an overview of Hepatitis B Virus (HBV) infection and non-radiolabel based detection methods for HBV DNA are described. Also included in this chapter are introductions to laser induced fluorescence, DNA separation in cellulose polymer solutions and single-cell analysis using CE.;In chapter 2, the use of hydroxyethyl cellulose (HEC) as a replaceable sieving polymer for DNA separation was described. Polymer concentration, separation efficiency, migration time, degradation of the sieving matrix were studied. A polymer concentration of 0.8% (w/v) was found to be suitable for the separation of a range of dye-labeled DNA fragments (229 to 903 bases) under denaturing conditions. HEC solution prepared in 0.1 M TAPS buffer is stable for at least 6 months at 4°C. This chapter also describes the construction of a CE instrument.;In chapter 3, a unique on-column labeling method for DNA fragments is described. In this technique, DNA fragments are labeled and detected simultaneously by interaction with oppositely migrating dye molecules during electrophoresis. Under this condition, significant amount of electrostatic interaction between the cationic dyes and the DNA fragments was demonstrated. This interaction could be preserved in the presence of excess dye in the system. The approach was able to resolve DNA fragments over a large size range (72bp to 23 kbp). The sensitivity of detection could be improved 4-fold by stacking dye molecules at the detector end of the capillary using a discontinuous buffering system.;Chapter 4 describes the detection of duck hepatitis B virus (DHBV) DNA using PCR and CE-LIF detection. A multi-primer system was used to demonstrate the reduction of DHBV DNA in serum and liver of infected ducklings upon antiviral treatment. The use of this approach did not demonstrate truncation of viral DNA in response to short duration of treatment with (-)-beta-L2 ',3'-dideoxy-3'-thiacytidine. The same experiment indicated that 2-amino-6-methoxy purine 2' 3' dideoxyriboside is not able to completely inhibit viral DNA synthesis by blocking the 5' end of the viral DNA strand, suggesting that a longer duration of treatment is necessary to completely inhibit synthesis of viral DNA.;In chapter 5, quantitative competitive (QC) PCR were modified to address the accuracy of quantification and to facilitate eventual automation of this technique. The modified technique was used to investigate the efficiency of amplification during PCR, we showed that differences in amplification efficiency between a target and a competitor limit the accuracy of quantification using end point measurement. These differences fluctuated from cycle to cycle, and tended to give rise to high discrepancies in quantification at high cycle numbers. Sampling of PCR products from a range of cycles in which amplification efficiency is a constant would reduce these discrepancies.;In chapter 6, studies of enzyme activity using CE-LIF are reported. This chapter emphasizes the construction of a CE instrument that is capable of single-cell analysis. Using baker's yeast as a model, in vivo enzymatic hydrolysis of a fluorescence-labeled sugar substrate was studied. We showed that several hundred molecules of enzyme reaction products could be detected in a single yeast spheroplast. Confocal laser scanning microscopy confirmed the uptake and internalization of fluorescent substrate.;Chapter 7 addresses the potential use of CE-LIF for the detection, and quantification of DNA. Future development of CE for medical diagnosis using a multichannel system for high throughput screening is suggested.