394

Electrophoresis 1992, 13, 394-395

.I.Noolandi

Short communications Jaan Noolandi Xerox Research Centre of Canada, Mississauga, Ontario

A new concept for sequencing DNA by capillary electrophoresis It is proposed that the scaling symmetry of constant charge densitywith increasing molecular weight, which prevents the separation by electrophoresis of DNA molecules in solution (with respect to molecular weight) be broken by the attachment of a perturbing entity (protein, virus or charged sphere) to one end of the molecule. An application of this idea to a concept for sequencing DNA by capillaryelectrophoresis is discussed, and the possibility of using the reattachment ofthe RecA protein to separate large segments of DNA in solution by electrophoresis following sequence-specific cleavage is mentioned.

The concept ofbroken symmetry in physics is useful for understanding the different ways of separating DNA by electrophoresis. For the gel electrophoresis of large DNA molecules in constant electric fields, the identical scaling with size of the electrical force on the stretched molecule and the opposing friction due to the separating medium (gel) can be broken by the use of pulsed electric fields, allowing the separation of megabase-sized molecules [I]. Similarly, the separation of large globular DNA by free flow electrophoresis is not possible because of the identical scaling of the electrical force on the uniform charge density of the DNA globule in solution, and the opposing friction due to the almost free drainage of the buffer through the polyelectrolyte [2]. Recently, multiwavelength fluorescence detection has made possible the sequencing of DNA by capillary electrophoresis [3], although difficulties with the separating medium in the capillary have limited the usefulness of this technique. Here we propose a novel method of sequencing by capillary electrophoresis in which a standard buffer solution is used, instead of a gel or a viscous medium such as a polymer solution [4-61, and the above-mentioned symmetry is broken by the attachment of each strand of DNA to be sequenced to one of a collection of charged spheres or proteins, which are highly monodisperse with respect to their charge/mass ratio. In the enzymatic method of sequencing, DNA fragments are synthesized by DNA polymerase, which incorporates deoxynucleotide monomers into a polymeric complementary copy of a template DNA fragment [7]. An oligonucleotide primer is used to initiate the synthesis of a new DNA strand from a specified location. In this technique four separate reactions are performed, each with all of the four deoxynucleotides but only one of the four dideoxynucleotides, which terminate the extension products at a specified base and produce a nested set of all fragments which end with that base. Here we propose using dideoxynucleotide analogs for each base which, as before, lack the chemical functionality for further chain elongation [8], but are modified at the sugar moiety by the attachment of a functional group R [9]. Figure 1 shows a nucleoside 5'-triphosphate, d-NTP (3'-R), which can be used for enzymatic chain termi-

Correspondence: Dr. Jaan Noolandi, Xerox Research Centre of Canada 2660 Speakman Drive, Mississauga, Ontario, Canada Abbreviation: ss, single-stranded

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nation instead of ddNTP as in the Sanger method [7]. This allows the enzymatically generated nested set of DNA fragments, each ending with a particular dideoxynucleotide and a functional group, to be attached individually to different substrates by means of a site with a corresponding functional group. Base - A,G,C,T

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A number of different kinds of polymerized surfactant vescicles are, in principle, available [lo-121 for attachment to single-stranded DNA (ssDNA). Many reactive moieties can be used to modify lipids to make them polymerizable, e.g., diacetylene, methacryloyl, dienoyl, sorbyl, styryl, vinyl, thiol and lipoyl. Polymerizable groups may be incorporated into one or both of the hydrophobic tails near the middle or at the end of the chain(s), and reactive groups may be attached to the hydrophilic head group or electrostatically associated with a charged 1ipid.The compatibility of the coupling conditions of the charged ball or protein with the structural integrity of the ssDNA is an important criterion in selecting the optimal chemical composition of the ball, and remains to be determined. Vescicles which are, as one specific example, composed of surfactant SorbPC molecules [lo] (shown in Fig. 2) and functionalized cosurfactant molecules with an end group R can be made with negative charges on the surface byneutralizing the positive charges on the SorbPC molecules before vescicle formation. The functional group indicated by R in Fig. 1 could be an N, group, and the partner in the coupling reaction with the end group R on the vescicle could be a carbon-carbon triple bond or a three-valent phosphorus compound [lI]. The vescicle structure can be fixed after formation by UV polymerization 112,131.The polymerized vescicles, which will generally have a distribution of sizes and charge/mass ratios, can be fractionated by capillary electrophoresis, using a high electric field (300-500 V/cm). Alternatively, one could use a commercially available We0173-0838/92/0606-0394 $3.80+.25/0

Electrophoresis 1992, 13, 394-395

Sequencing DNA by capillary electrophoresis

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ber-Holbein free flow apparatus for this preparative step, which would have the advantage that much larger amounts of material could be fractionated. Once a sufficiently monodisperse fraction of vescicles has been obtained, they can be exposed to the single strands of DNA with functional groups attached, as prepared previously. With the proper concentrations of reactants, most of the vescicles will react through a functional group with only one chain.Those with more than one attached chain will be distinguishable during electrophoresis by their anomalous mobilities. Yet another possibility for choosing monodisperse charged “partic1es”is to use a protein or charged virus, in which case the charge/mass ratio is identical for each particle. However, the aggregation of these entities during the attachment reaction step to ssDNA may be a problem. The different length DNAfragments, with electricallyindentical vescicles attached, can be separated by capillary electrophoresis using a buffer which solubilizes the “ball and chain” complexes. Sequencing can be carried out by using fluorescent primer chemistry for the detection of the fragments ending with a given base [3]. The advantage of this method is an increase in throughput (in the number of bases sequenced per clone per hour) by an estimated several orders of magnitude, since it should be possible to sequence several hundred bases in minutes or seconds instead of hours by using capillary electrophoresis with a high electric field. The accuracy of the method will also likely be higher than with current techniques, since a standard buffer solution can be used, instead of a gel or a viscous medium [3-61. However, the resolution of the proposed technique is highly dependent on the monodispersity of the mobility of the charged vescicles, since otherwise one cannot distinguish vescicles with different charge/mass ratios pulling DNA fragments of the same length from those with the same charge/mass ratios but with different lengths of attached DNA single strands. The fractionation of the charged, functionalized vescicles by capillary or free flow electrophoresis before reaction with the single-stranded, functionalized DNA strands is an important step in the proposed method. The size of the vescicles is determined by the molecular weight of the SorbPC molecule, and is to be chosen according to the resolution required in sequencing by capillary electrophoresis, as well as the monodispersity required in the fractionation of the charged vescicles before DNA attachment. However, the use of dideoxynucleotide analogs for attaching single strands of DNA to functionalized vescicles is not the only possibility for assembling “ball and chain” complexes. Recent advances in streptavidin-biotin technology

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Figure 3. Proposed separation of nested set of single-stranded DNAfragments by capillary electrophoresis, using pulsed or constant electric fields, with monodisperse (chargdmass ratio) vescicles or proteins attached to the functionalized dideoxynucleotide analog which terminates all the fragments at base A (in the above example) and with a fluorescent primer at the 3’ end of each fragment.

[ 141,using commercially available small beads, afford other possibilities for attaching DNA to charged spheres, although it is not known at present whether the currently available sizes and chargelmass ratios are well-suited for the sequencing by capillary electrophoresis application. Also, the attachment of synthetic polyelectrolytes of different lengths to charged, monodisperse beads gives an interesting system for which theoretical models of hydrodynamic flow can be compared with the mobilities measured by capillary electrophoresis. Finally, the reattachment of the RecA protein following selective cleavage of human DNA using RecA assisted restriction endonuclease (RARE) cleavage [15] may allow the separation of the resulting large duplex DNA segments by electrophoresis in solution. Received December 3. 1991

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