Polyacrylamide solutions for DNA sequencing by capillary electrophoresis: mesh sizes, separation and dispersion.
Two preparations of linear polyacrylamide with average molecular weights of 0.37 million and 1.14 million Da, and a deuterated preparation with an average molecular weight of 1.71 million Da, were used to study the effects of molecular weight, polydispersity, and concentration on the mesh size of entangled polymers in a DNA sequencing buffer solution and their ability to resolve DNA sequencing reactions by capillary electrophoresis. The polyacrylamide concentrations were above the overlap threshold C*, the concentration above which an entangled polymer network is expected to form. Small angle neutron scattering experiments showed that between 1% and 8% polyacrylamide, the mesh size ( xi ) can be expressed by the relation xi = 2.09C-0.76, where xi is in A and C is the polymer concentration in g/mL. The mesh size depended only on the concentration and was independent of the average molecular weight of the polyacrylamide. Consistent with this result, electrophoretic mobilities of DNA moving through the polymer network depended almost entirely on the polyacrylamide concentration and not on its molecular weight or polydispersity. Although separation was little affected, band sharpness persisted to longer DNAs when the polymer network contained a higher fraction of larger polyacrylamide molecules. We postulate a dispersive effect that depends on the size of the DNA and the resiliency of the polymer network. This interpretation provides a rationale for optimizing the design of polymer solutions to sieve DNA for sequencing by capillary electrophoresis.
Wu
C
C
Department of Chemistry, State University of New York at Stony Brook 11794-3400, USA.
Quesada
M A
MA
Schneider
D K
DK
Farinato
R
R
Studier
F W
FW
Chu
B
B
D000180:Acrylic Resins / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels / Q000737:chemistry*; D008970:Molecular Weight; D017422:Sequence Analysis, DNA; D012996:Solutions
D000180:Acrylic Resins; D004277:DNA, Single-Stranded; D005782:Gels; D012996:Solutions; C016680:polyacrylamide gels