A SAMPLE PURIFICATION METHOD FOR RUGGED AND HIGH-PERFORMANCE DNA-SEQUENCING BY CAPILLARY ELECTROPHORESIS USING REPLACEABLE POLYMER-SOLUTIONS - A - DEVELOPMENT OF THE CLEANUP PROTOCOL

A method for the cleanup of Sanger DNA sequencing reaction products fo r capillary electrophoresis analysis with replaceable polymer solution s has been developed. A poly(ether sulfone) ultrafiltration membrane p retreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin colum n format (two columns per sample) was employed to decrease the concent ration of salts below 10 mu M in the sample solution. The method was v ery reproducible and increased the injected amount of the sequencing f ragments 10-50 fold compared to traditional cleanup protocols. Using M 13mp18 as template, the resulting cleaned-up single DNA sequencing fra gments could routinely be separated to more than 1000 bases with a bas e-calling accuracy of at least 99% for 800 bases. The method is simple and universal and can be easily automated. In the following paper, a systematic study to determine quantitatively the effects of the sample solution components such as high-mobility ions (e.g., chloride and di deoxynucleotides) and template DNA on the injected amount and separati on efficiency of the sequencing fragments is presented.