Zhou Ping, Yu Shengbing, Liu Zhonghua, Hu Jiming, Deng Yanzhuo
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
J Chromatogr A. 2005 Aug 12;1083(1-2):173-8. doi: 10.1016/j.chroma.2005.05.096.
A new separation matrix, consisting of polymer poly(N-isopropylacrylamide) (PNIPAM) and small molecule additive mannitol, was used for double-stranded (ds) DNA and plasmid DNA separation by capillary electrophoresis. The matrix had a low viscosity, which made it very easy to handle. The additive mannitol dramatically enhanced the sieving performance of PNIPAM in TBE buffer. The optimal mannitol concentration 6% in polymer solution, was determined with the consideration of both speed and resolution. A resolution of 0.95 was achieved on the separation of 271/281 bp in the phiX174/HaeIII digest by using 1.5% PNIPAM + 6% mannitol, while the supercoiled, linear and nicked conformers of lambda plasmid were separated in 1% PNIPAM + 6% mannitol, demonstrating the potential use of this new matrix for effective DNA separations. The dramatic impact of mannitol on sieving performance of PNIPAM solution was investigated. pH dependent self-coating ability of PNIPAM was revealed. The presence of mannitol in TBE buffer decreased the pH of the buffer, which led to more efficient self-coating ability of PNIPAM probable due to the formation of hydrogen bonds between PNIPAM molecules and silanol groups at the silica wall.
一种由聚合物聚（N-异丙基丙烯酰胺）（PNIPAM）和小分子添加剂甘露醇组成的新型分离基质，被用于通过毛细管电泳分离双链（ds）DNA和质粒DNA。该基质具有低粘度，这使其非常易于处理。添加剂甘露醇显著提高了PNIPAM在TBE缓冲液中的筛分性能。考虑到速度和分辨率，确定了聚合物溶液中甘露醇的最佳浓度为6%。通过使用1.5% PNIPAM + 6%甘露醇，在phiX174/HaeIII消化物中分离271/281 bp时实现了0.95的分辨率，而在1% PNIPAM + 6%甘露醇中分离了λ质粒的超螺旋、线性和切口构象体，证明了这种新型基质在有效DNA分离方面的潜在用途。研究了甘露醇对PNIPAM溶液筛分性能的显著影响。揭示了PNIPAM的pH依赖性自涂层能力。TBE缓冲液中甘露醇的存在降低了缓冲液的pH值，这可能由于PNIPAM分子与硅壁上的硅醇基团之间形成氢键而导致PNIPAM具有更有效的自涂层能力。
