Polyacrylamide solutions for DNA sequencing by capillary electrophoresis: Mesh sizes, separation and dispersion

Abstract

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.

Capillary zone electrophoresis of proteins in semidilute polymer solutions: Inter- and intra-polymer predictability of size-dependent retardation

“…Yet, this interaction appears to be size-dependent as well ( [18,19] and Tables 3 and 4), thus ruling out hydrophobic interactions or hydrogen bonding which would depend on the exposure of specific sites on the protein surface rather than protein size. A further problem is that, despite the good matching between k PA and k* PA , they both sufficiently exceed the k value for aqueous solutions of polyacrylamide, obtained by small angle neutron scattering experiments [27], viz. k % 0.21 [x(nm) = 0.21 c ±0.76 ].…”

Polymer solutions and entropic‐based systems for double‐stranded DNA capillary electrophoresis and microchip electrophoresis

“…Although both manufacturing procedures are similar, LPA is less prone to formation of bubbles. LPA solution provides the longest ssDNA read length unsurpassed by other linear or branched polymer solutions [74][75][76], and has been the workhorse matrix in DNA sequencing. Unfortunately, LPA has no self-coating ability, so it has to be used in precoated separation channels.…”

Nanostructured polymer matrix for oligonucleotide separation

“…The oligonucleotide sample was electrokinetically injected into the capillary at an electric field of 300 V/cm for 1 s. The running voltage was 200 V/cm. The CE setup has been described elsewhere [31].…”