‘Laterally aggregated’ polyacrylamide gels for electrophoresis

Abstract

A new method is described for producing highly porous polyacrylamide matrices: polymerization in presence of a preformed hydrophilic polymer. If a standard mixture of monomers (e.g., 5%T, 4%C) is polymerized in presence of, e.g., polyethylene glycol (PEG) 10 kDa, lateral chain aggregation occurs, with formation of large pore sizes. In PEG 10 kDa, the transition from a small- to a large-pore gel is clearly apparent at 0.5% PEG addition and reaches a plateau already at 2.5% PEG. Even with shorter PEG fragments (6.2 and 1 kDa) this transition occurs, but with progressively larger amounts of PEG in solution (up to 25% for the 1 kDa species). Other polymers such as hydroxymethyl cellulose (1000 kDa) and polyvinyl-pyrrolidone (360 kDa and 25 kDa) are also able to elicit this phenomenon. It appears that lateral chain aggregation (before the cross-linking event) is induced via intra-chain hydrogen bonding, since urea and temperature strongly inhibit it, whereas tetramethylurea (an agent quenching hydrophobic interactions) does not hamper it. By scanning electron microscope, it is found that the maximum pore size obtained in a 5%T, 4%C gel in presence of 2.5% PEG 10 kDA is of the order of 0.5 micron, whereas the same 5%T, 4%C control gel would have an average pore diameter of 5 nm. Thus, an increment of pore size of about 2 orders of magnitude is obtained: in these new matrices, a 21000 bp DNA fragment exhibits a much greater migration than in a control gel in which the sample is entrapped at the application site.

Photopolymerization of polyacrylamide gels with methylene blue

“…mmol/mL). In some experiments, persulfate/ TEMED initiated gels were polymerized in presence of a 'laterally-aggregating agent', 2.5% polyethylene glycol (PEG) 10 000, to produce macroporous structures [15].…”

Towards new formulations for polyacrylamide matrices: N‐acryloylaminoethoxyethanol, a novel monomer combining high hydrophilicity with extreme hydrolytic stability

“…20-30 nm in highly diluted matrices [7]. This limits the use of polyacrylamides for protein separations, whereas agarose gels are today almost exclusively used for separation of nucleic acid fragments.…”

Fluorescent in situ sequencing on polymerase colonies

“…It is possible that the maximum length of an extension product may be limited because the polymerase is trapped in an acrylamide pore. However, a 34-bp extension corresponds to a 11.9-nm translocation of the DNA polymerase on the DNA Green S1 CACACA CACACA T1: CACACACACACACACTC 2 0 Red S2 GTGT GTGT T2: GTGTGTGTGTGTGTGTC 4 0 Lt blue S3 AGTGC AGTGC T3: AGTGCTCACACACGTGATC 3 0 Dk blue S4 CAGCGA CAGCGA T4: CAGCCGAACGACCGATC 5 0 Yellow S5 AGTGT ATGT T5: ATGTGAGAGCTGTCGTC 4 1 template, a length greater than the predicted pore size (2 nm) of a 15% acrylamide gel with 5% cross-linker [27]. Therefore, it seems that acrylamide pore size does not limit FISSEQ read length in the useful range in which we are working, possibly because of polymer flexibility.…”