The impact of a plug of salts on the analysis of large volumes of dsDNA by capillary electrophoresis.
A partially filling technique for the analysis of DNA markers and polymerase chain reaction (PCR) products by capillary electrophoresis in the presence of electroosmotic flow using polymer solutions is presented. Either after or prior to the sample injection, a plug of salts at high pH was hydrodynamically injected. During the separation, poly(ethylene oxide) (PEO) solution entered the capillary. We have found that the position, length, and composition of the plugs affect the sensitivity, resolution, and speed on the analysis of PhiX-174/HaeIII DNA restriction fragments or a DNA mixture (pBR 322/HaeIII digest, pBR 328/BglI digest and pBR 328/HinfI digest) with different degrees. Through careful evaluation of the impact of anions and cations on the analysis of DNA, we have suggested that the optimal condition is applying a plug consisting of 32 mM NaCl and 0.01 M NaOH at 30 cm height for 60 s after sample injection. In the presence of such a plug, PEO adsorption reduces, and thus the separation is faster, as well as the sensitivity improves. Using this condition, the analysis of a DNA mixture (injected at 30 cm for 360 s) containing ten different PCR products amplified after 17 cycles was complete in 25 min. About a 2000-fold improvement in the sensitivity was achieved when compared to that by a conventional method (10 s injection) without applying a plug.
Hsieh
Ming-Mu
MM
Department of Chemistry, National Taiwan University, Taipei, Taiwan, ROC.
Chang
Po-Ling
PL
Chang
Huan-Tsung
HT
D004247:DNA / Q000032:analysis* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D011092:Polyethylene Glycols; D016133:Polymerase Chain Reaction; D012492:Salts
D012492:Salts; D011092:Polyethylene Glycols; D004247:DNA