Temperature-dependence of preconditioning for lengthened capillary DNA sequencing.
A previous study shows that electrophoretic preconditioning of a commercial polymer solution increases the spacing and resolution of DNA fragments fractionated in this solution by CE at 50 degrees C (Griess, G. A. et al., Electrophoresis 2005, 26, 102). The present study shows that this preconditioning effect on peak spacing progressively increases when the temperature of preconditioning increases to 70 degrees C, though fractionation is still performed at 50 degrees C. An increase in peak sharpness accompanies the increase in peak separation for DNA fragments longer than 200 bases. Changing the preconditioning temperature from 50 to 70 degrees C optimally improves resolution of fragment analysis in the range of 600-2000 nucleotides. When DNA sequencing is performed with automated base calling and 70 degrees C preconditioning at 319 V/cm (47 cm long capillary, Applied Biosystems 310 apparatus), the range of high-quality base calls is increased by 25% to 750; the range of low-quality base calls is increased by about 100% to 1200 in comparison to DNA sequencing without preconditioning.
Griess
Gary A
GA
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.
Hardies
Stephen C
SC
Serwer
Philip
P
D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D010957:Plasmids / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature
D004247:DNA