Madabhushi R S
Perkin Elmer Corporation, Applied Biosystems Division, Foster City, CA, USA.
Electrophoresis. 1998 Feb;19(2):224-30. doi: 10.1002/elps.1150190215.
A low viscosity (ca. 75cP) solution using polydimethylacrylamide (PDMA) was developed for separating DNA sequencing extension products by capillary electrophoresis (CE). This medium gave a length-of-read (LOR) value of approximately 600 bases in about 2 h using four-color sequencing in 50 microm capillary at 42 degrees C under a field of 160 V/cm. This medium also works in bare capillaries by noncovalently coating the surface to suppress both electroosmotic flow (EOF) and DNA-capillary wall interactions, and eliminates the need for complicated covalent coatings. At least 100 successive sequencing runs were performed in the same capillary by simply pumping fresh medium after every run, without requiring any reconditioning of the capillary surface between runs. The thermal stability of the noncovalent coating can be improved by adding small amounts of high molecular weight PDMA to the separation medium. The advantages of low viscosity separation media and uncoated capillaries are of paramount importance to develop high-throughput instruments for DNA sequencing.
开发了一种使用聚二甲基丙烯酰胺（PDMA）的低粘度（约75厘泊）溶液，用于通过毛细管电泳（CE）分离DNA测序延伸产物。在42摄氏度、160 V/cm的电场下，使用50微米毛细管进行四色测序时，这种介质在约2小时内可获得约600个碱基的读长（LOR）值。这种介质还可在裸毛细管中工作，通过非共价包被表面来抑制电渗流（EOF）和DNA与毛细管壁的相互作用，并且无需复杂的共价包被。通过每次运行后简单地泵送新鲜介质，在同一毛细管中至少进行了100次连续测序运行，运行之间无需对毛细管表面进行任何重新处理。通过向分离介质中添加少量高分子量PDMA，可以提高非共价包被的热稳定性。低粘度分离介质和未包被毛细管的优点对于开发用于DNA测序的高通量仪器至关重要。
