A Sample Purification Method for Rugged and High- Performance DNA Sequencing by Capillary Electrophoresis Using Replaceable Polymer Solutions. B. Quantitative Determination of the Role of Sample Matrix Components on Sequencing Analysis

摘要

In the previous paper, a sample cleanup procedure for DNA sequencing reaction products was developed, in which template DNA was removed by ultrafiltration and the total concentration of salts (chloride and di- and deoxynucleotides) was decreased below 10/tM using gel filtration. In this paper, a quantitative study of the effects of these sample solution components on the injected amount and separation efficiency of the sequencing fragments in capillary electrophoresis is presented. The presence of chloride and deoxynucleotides in a total concentration above 10/μM in the sample solution significantly decreased the amount of DNA sequencing fragments injected into the capillary column. However, the separation efficiency was not affected upon increasing the amount of salt. On the other hand, in the presence of only 0.1μg of template in the sample (one-third of the lowest quantity recommended in cycle sequencing) and at very low chloride concentration (~5/μM), the separation efficiency decreased by 70%, and the injected amount of DNA sequencing fragments was 40% lower compared to the sample cleaned by the new purification method. The deleterious effect of template DNA on the separation of sequencing fragments was suppressed in the presence of salt in a concentration above 100 /μM in the sample solution. Separately, it was found that both the electric field strength and duration of injection affected the resolution of DNA sequencing fragments when the cleaned up sample solution was used. Separation efficiencies of 15 × 106 theoretical plates/m were achieved when the sample was loaded at low electric field, e.g., 25 V/cm for 80 s or less. The results demonstrate that the sample solution components (chloride, deoxynucleotides, template DNA) and injection conditions must be controlled to achieve high performance and rugged DNA sequencing analysis.