Electrophoresis zyxwvutsrqponmlkji 1996, zyxwvutsrqpon 17, 1451-1459 Flowable networks for zyxwv DNA sequencing by CE 1451 Flowable networks as DNA sequencing media in capillary columns Steve Menchen' Ben Johnson' Mitchell A. Winnik' Bai Xuz 'Perkin Elmer Corporation, Applied Biosystems Division, Foster City, CA, USA 'Department of Chemistry, University of Toronto, Toronto, Canada A novel class of materials that self-assemble in water into equilibrium network structures with a well-defined mesh size consist of polyethylene glycols (PEG'S) end-capped with micelle-forming fluorocarbon tails. These micellar systems form flowable aqueous gel-like networks that permit electrophoretic DNA sequencing in capillary columns. The gels have unusual rheological prop- erties, including network breakdown under shear, resulting in plug flow that allows colums refill with complete ejection of byproducts of the previous sequencing analysis. In this system, DNA fragment electrophoretic mobilities are unaffected by the hydrophobicity of the polymer tails. Low molecular weight (M) PEG chains zyxwvu (M 8000) show catastrophic resolution loss for DNA fragments larger than 100 bases due to band broadening. For a longer PEG segment (M 35 000) separating the end groups, band broadening occurs for DNA fragments larger than 300 bases, implying that the PEG segment length controls the mesh size in the equilibrium network structure. Optimum sequen- cing results were obtained from a 6% solution of a 1:l mixture of C,F,, end- capped- and C,F,, end-capped PEG 35 000. The resolution limit of fluorescent- dye-labeled sequencing products in this formulation was 450 bases in 75 pm capillaries at 200 V/cm. 1 Introduction Two strategies are currently used for the sieving com- ponent of DNA sequencing separation media. In slab format, gel electrophoresis on chemically cross-linked polyacrylamide gels (PAGE) provides high performance and substantial dynamic range [I] with resolution of polynucleotide fragments from 30 to 800 bases, differing in length by a single base, in a single experiment [2-51. PAGE gels have been successfully used for DNA sequencing with capillary electrophoresis (CE) [6-81, but these systems cannot be refilled after each experiment. Recently, flowable solutions of uncross-linked entangled linear water-soluble polymers have been demonstrated for CE DNA sequencing [9]. Both of these techniques rely on the intrinsic "mesh size" (tern) of the sieving polymer for DNA separation [lo, 111. This size is a func- tion of the polymer chain concentration (c,,,) and stiff- ness [12] zyxwvutsrq (*). The effective mesh size of a chemically cross-linked polymer depends on both the amount of cross-linker added to the reaction mixture and the detailed kinetics of the polymerization reaction [13]. For a linear, entangled polymer system, Teff depends on the polymer overlap concentration (c*), which is a function of the molecular weight (At) of the polymer [12]. The largest change of mesh size with cpo, occurs from zyxwvu dc to about 5 times dc (**); we have found that the high reso- lution performance required for DNA sequencing appli- cations normally involves cpo, > 5c*, a regime where changes in Sen with concentration are quite small. The process of selecting of a particular polymer for an entan- gled polymer sequencing medium can be guided by the ability to control Ser within the parameters of cP,,, polymer molecular weight, and polymer chain stiffness (persistence length) [12]. Since cpol > 5P is a desirable regime to perform DNA sequencing separations, only limited control of mesh size is possible by varying cpo,. It is more effective to increase chain length, thus lowering dc [12], but large molecular weight changes are required. For example, poly(ethy1ene oxide) (PEO) of M 600000 was found to give useful separation up to about 100 bases, but to get resolution to 400 bases, PEO of M 8 X lo6 was required [14]. The most effective method for altering Sen is to use a polymer with a larger persistence length; for example, linear polyacrylamide, a polymer with a much stiffer backbone than polyethylene glycol (PEG) , can be used for DNA sequencing in capillaries at M 100000 [9]. Here we describe an alternative strategy for making flow- able networks with an appropriate DNA sequencing mesh size that is based on self-assembled gels. A self- assembled gel refers to an aqueous solution of hydro- phobically modified water-soluble polymer that is deriva- tized in such a way that the hydrophobic entities undergo intermolecular aggregation creating a highly viscous system. A popular copolymer architecture of these gels is a central linear water-soluble polymer bearing hydrophobic end groups (telechelic polymer). These types of telechelic polymers are employed in the paint industry as rheology modifiers. One of their key characteristic features is that they show classical Newto- Correspondence: Dr. Steve Menchen, Perkin Elmer Corporation, Applied Biosystems Division, 850 Lincoln Center Drive, Foster City, CA 94404, USA (Tel: +415-638-5609; Fax: +415-572-2743) Nonstandard abbreviations: FWHM, full width at half maximum; PEG, polyethylene glycol Keywords: DNA sequencing / Capillary electrophoresis / Flowable net- works / Polyethylene glycol (*) From the relationship of c* = u-~N-'.' , where the persistence length a is a measure of polymer backbone stiffness, and N is the number of monomer units constituting the polymer chain [12]. (**) This is readily seen from the relationship €& = k (c,,,~)-'~~~, where k is a constant related to a particular polymer structure in solu- tion and is a function on the polymer persistance length a; and cpol is the polymer concentration above c* and is a function of the volume fraction of polymer in solution, @. This equation is derived from F(@) = [12]. zyx 0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1996 0173-0835/96/0909-1451 $10.00+.25/0