DNA sequencing of close to 1000 bases in 40 minutes by capillary electrophoresis using dimethyl sulfoxide and urea as denaturants in replaceable linear polyacrylamide solutions.
The goal of this work was to reduce the capillary electrophoresis (CE) separation time of DNA sequencing fragments with linear polyacrylamide solutions while maintaining the previously achieved long read lengths of 1000 bases. Separation speed can be increased while maintaining long read lengths by reducing the separation matrix viscosity and/or raising the column temperature. As urea is a major contributor to the separation buffer viscosity, reducing its concentration is desirable both for increase in the separation speed and easier solution replacement from the capillary. However, at urea concentrations below 6 M, the denaturing capacity of the separation buffer is not sufficient for accurate base-calling. To restore the denaturing properties of the buffer, a small amount of an organic solvent was added to the formulation. We found that a mixture of 2 M urea with 5% v/w of dimethyl sulfoxide (DMSO) resulted in 975 bases being sequenced at 70 degrees C in 40 min with 98.5% accuracy. To achieve this result, the software was modified to perform base-calling at a peak resolution as low as 0.24. It is also demonstrated that the products of thermal decomposition of urea had a deleterious effect on the separation performance at temperatures above 70 degrees C. With total replacement of urea with DMSO, at a concentration of 5% v/w in the same linear polyacrylamide (LPA)-containing buffer, it was possible to increase the column temperature up to 90 degrees C. At this temperature, up to 951 bases with 98.5% accuracy could be read in only 32 min of separation. However, with DMSO alone, some groups of C-terminated peaks remained compressed, and column temperature at this level cannot at present be utilized with existing commercial instrumentation.
Kotler
Lev
L
Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA 02115, USA.
He
Hui
H
Miller
Arthur W
AW
Karger
Barry L
BL
D000180:Acrylic Resins; D002021:Buffers; D004247:DNA / Q000032:analysis / Q000737:chemistry; D004121:Dimethyl Sulfoxide; D019075:Electrophoresis, Capillary / Q000379:methods*; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods; D012996:Solutions; D013997:Time Factors; D014508:Urea; D000099166:Polyacrylamides
D000180:Acrylic Resins; D002021:Buffers; D004247:DNA; D004121:Dimethyl Sulfoxide; D012996:Solutions; D014508:Urea; D000099166:Polyacrylamides