Capillary gel electrophoresis and DNA.

摘要

Capillary gel electrophoresis (CGE) is a rapid method of separation of biological samples. Promising applications of CGE include the separation of DNA sequencing samples (ssDNA) and double-stranded DNA (dsDNA). CGE allows faster separation of DNA with equal or higher resolution versus conventional polyacrylamide gel electrophoresis (PAGE). In CGE the samples are separated in series, thus the gel-filled capillary is used for a few subsequent separations in order to reduce the experimental time. However, the reproducibility and the stability of CGE is a major problem.;The first step to do before studying the reproducibility of CGE is to find the optimum polyacrylamide concentration. The total gel concentration (%T) has been studied previously. In Chapter 2, the effect of the concentration of cross-linker (%C) in polyacrylamide on the separation of ssDNA is studied. High %C gives higher resolution with longer separation time than 0%C. The number of fragments that can be separated seems to increase as the %C decreases. This means that 0 %C polyacrylamide would be more useful than high %C polyacrylamide for DNA sequencing applications.;In chapter 3, the stability of 5%T 0%C polyacrylamide is studied by looking at the effects that aging polyacrylamide, and buffer transference numbers have on the conductivity of polyacrylamide. It is shown that by using aged polyacrylamide (6 days or more), more stable conductivity profiles are obtained.;In Chapter 4, we look at the effect that the stabilization of the conductivity of polyacrylamide has on the separation of ssDNA. First, it is demonstrated that as the polyacrylamide ages (i.e. yielding more stable conductivity) the migration times of the DNA fragments for subsequent sequencing runs are more stable. Second, stable multiple sequencing runs are reported by reversing the polarity for a short period of time between each run.;In the first part of Chapter 5, dsDNA are labeled with the novel intercalating dyes YOYO-1, YOYO-3, and POPO-3 and separated on polyacrylamide and hydroxypropylmethylcellulose. The limit of detection (3