Plant & Animal Genomes XIII Conference

January 15-19, 2005
Town & Country Convention Center
San Diego, CA

High Throughput DNA Analysis By Multiplexed Constant And Pulsed Field Capillary Electrophoresis With UV Absorbance Detection

Wei Wei , Jeremy Kenseth , Dennis Tallman

  CombiSep, 2711 South Loop Drive, Suite 4200, Ames, IA 50010, USA

The separation and sizing of DNA fragments has widespread application in genomics. This poster will describe a new strategy for sizing and quantification of DNA fragments across a wide size range by multiplexed capillary gel electrophoresis (CGE) with UV absorbance detection. Variations in migration times and UV absorbance response in different capillaries are normalized through the use of two internal standards. Unknown DNA fragments can be sized and quantified with high accuracy when incorporating a standard sizing ladder in one capillary of the array and bracketing with the internal standards in capillaries containing the unknown samples. Two different separation matrices have been developed to resolve PCR products ranging from 100 bp to 2 kbp and DNA fingerprint fragments from 2 kbp to 20 kbp. These new matrices have low viscosity and are compatible with uncoated capillaries. We also first demonstrate multiplexed CGE with pulsed field electrophoresis, which can provide very high separation efficiency for larger DNA fragments. In addition, as salt removal from the sample matrix is essential for successful and reproducible DNA separations by CGE, a protocol for high throughput product desalting will be described.