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

In the previous paper, a sample cleanup procedure for DNA sequencing r eaction products was developed, in which template DNA was removed by u ltrafiltration and the total concentration of salts (chloride and di- and deoxynucleotides) was decreased below 10 mu M using gel filtration . In this paper, a quantitative study of the effects of these sample s olution components on the injected amount and separation efficiency of the sequencing fragments in capillary electrophoresis is presented. T he presence of chloride and deoxynucleotides in a total concentration above 10 mu M in the sample solution significantly decreased the amoun t of DNA sequencing fragments injected into the capillary column. Howe ver, the separation efficiency was not affected upon increasing the am ount of salt. On the other hand, in the presence of only 0.1 mu g of t emplate in the sample (one-third of the lowest quantity recommended in cycle sequencing) and at very low chloride concentration (similar to 5 pM), the separation efficiency decreased by 70%, and the injected am ount of DNA sequencing fragments was 40% lower compared to the sample cleaned by the new purification method. The deleterious effect of temp late DNA on the separation of sequencing fragments was suppressed in t he presence of salt in a concentration above 100 mu M in the sample so lution. Separately, it was found that both the electric held strength and duration of injection affected the resolution of DNA sequencing fr agments when the cleaned up sample solution was used. Separation effic iencies of 15 x 10(6) theoretical plates/m were achieved when the samp le was loaded at low electric field, e.g., 25 V/cm for 80 s or less. T he results demonstrate that the sample solution components (chloride, deoxynucleotides, template DNA) and injection conditions must be contr olled to achieve high performance and rugged DNA sequencing analysis.