Separation of Small DNAs by Gel Electrophoresis in a Fused Silica Capillary Coated with a Negatively Charged Copolymer

Abstract

Active development of compact analytical instruments suitable for point-of-care testing (POCT) requires optimization of existing methods. To aid the development of capillary gel electrophoresis instruments for POCT, we attempted to separate polymerase chain reaction products (small DNAs) using a short, fused silica capillary coated with an acrylamide (AM)/acrylic acid (AA) copolymer (poly(AM-co-AA)). To realize the high capability of this capillary to separate small DNAs, the magnitude of electroosmotic flow (EOF) was controlled by varying the content of negatively charged AA in the copolymer, which significantly affected the separation ability. At an AA content ≥3.75 mol %, sample DNAs could not be injected into the copolymer-coated capillary owing to strong EOF, whereas a 100 bp DNA ladder sample was successfully separated at an AA content of ≤3.5 mol %, showing that even slight AA content variations impact DNA flow. EOF values measured using a neutral coumarin 334 solution suddenly decreased at an AA content of 3.5 mol % relative to those at an AA content of ≥3.75 mol %. Theoretical plate values revealed that an AA content of 2.75 mol % was optimal for separating ladder DNAs with sizes ≥600 bp. Hence, EOF control achieved by varying the amount of negatively charged AA in the poly(AM-co-AA) coating can promote further development of short capillaries for POCT applications.