Detection of duck hepatitis B virus DNA fragments using on-column intercalating dye labeling with capillary electrophoresis-laser-induced fluorescence

A rapid on-column DNA labeling technique is used to detect viral restrictio n DNA fragments by capillary electrophoresis-laser induced fluorescence det ection. Intercalating dyes such as POPO3 or ethidium homodimer-2 are incorp orated into the detection buffer. The cationic dyes migrate into the capill ary during electrophoresis and bind to the oppositely migrating DNA fragmen ts. A post-column sheath-how fluorescence detector is used in the experimen t. Excellent labeling efficiency is achieved at minimal background fluoresc ence by diluting the dyes to between 1.10(-7) M and 5.10(-7) M in a buffer with low ionic strength relative to the running buffer within the capillary . This dilute sheath-flow buffer allows stacking of dye molecules inside th e capillary when an electric field is applied. Calibration curves using a s eries of DNA size markers (between 72 and 1353 base pairs) were linear over an order of magnitude in DNA concentration. Sensitivity also increased lin early with fragment length, and detection limits ranged from 4.10(-14) M to 5.10(-13) M for the size-standards. Analysis of cloned viral DNA using duc k hepatitis B virus demonstrated a concentration detection limit of 3.9.10( -16) M. Last, the technique produced very high separation efficiency, 14.10 (6) theoretical plates which is greater than 47.10(6) plates m(-1), for the duck hepatitis B viral genome. (C) 1999 Elsevier Science B.V. All rights r eserved.