Nucleic acid separation and detection by electrophoresis with a counter-migrating high-affinity intercalating dye

A method for separating and detecting nucleic acids contained in a sample by Capillary Electrophoresis, comprising:

• (a) providing a capillary tube having an anode end and a cathode end, wherein the capillary tube is filled with a high-resolution buffer, wherein the high-resolution buffer comprises a high-resolution intercalating dye, wherein the high-resolution intercalating dye is selected from group consisting of Ethidium Bromide (EB), Propidium Iodide (PI), and Acidine Orange (HCl salt);
• (b) loading the sample into the capillary tube from the cathode end;
• (c) separating the nucleic acids contained in the sample by applying a voltage between the anode and the cathode to move the nucleic acids from the cathode end toward the anode end in the presence of the high-resolution buffer;
• (d) loading a high-affinity intercalating dye into the capillary tube from the anode end, wherein the intercalating dye has a positive charge and DNA affinity constant of at least 106M−1, and wherein the intercalating dye moves from the anode end toward the cathode end under the voltage applied therebetween, and forms complexes with the separated nucleic acids;
• and
• (e) detecting the complexes.

A method for separating and detecting nucleic acids contained in a sample by CE-LIF with multiple intercalating dyes, comprising:

• (a) providing a capillary tube having an anode end and a cathode end, wherein the capillary tube is filled with a gel buffer comprising a high-resolution intercalating dye, wherein the high-resolution intercalating dye is selected from a group consisting of Ethidium Bromide (EB), Propidium Iodide (PI), and Acidine Orange (HCl salt);
• (b) loading the sample into the capillary tube from the cathode end;
• (c) separating the nucleic acids contained in the sample by applying a voltage between the anode and the cathode to move the nucleic acids from the cathode toward the anode in the presence of the high-resolution buffer;
• (d) loading a high-affinity intercalating dye into the capillary tube from the anode end, wherein the intercalating dye has a positive charge and DNA affinity constant of at least 106M−1, and wherein the intercalating dye moves from the anode end toward the cathode end under the voltage applied therebetween, and forms complexes with the separated nucleic acids;
• and
• (e) detecting the complexes.