Anal. Chem. I@91,63,2795-2797 (62) . . Yao, T.; Kobayashl, N.; Wasa, T. Anal. CMm. Acta 1990, 231, 121-124 (63) Yao, H,; Wasa, T, Anal. chkn.Acta 1990, 236, 437-440.
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Research (Y.U.) from the Ministry of Education, Science, and Culture, Japan, by the Japan-U.S. Cooperative'sciente'program (Y.U., T.K.) sponsored by the Japan Society for the Promotion of Science and the United States Nation2 Science Foundation, and by the United States Army Research Office Grant DAAL 03-88-K-0017 (E.K.M.).
T.;.Yaimto,
RECEIVEDfor review June 14,1991. Accepted September 12, 1991. This work is supported by a Grant-in-Aid for Scientific
TECHNICAL NOTES Improved Separation of Nucleic Acids with Analyte Velocity Modulation Capillary Electrophoresis Tshenge Demana, Maureen Lanan, and Michael D. Morris* University of Michigan, Department of Chemistry, Ann Arbor, Michigan 48109-1055
INTRODUCTION Capillary gel electrophoresis (CGE) is increasingly applied to the separation and sequencing of nucleic acids. The separation of polydeoxyoligonucleotides (l),single-stranded DNA sequence reaction products (2), as well as double-stranded DNA fragments (3, 4 ) , have been reported. As with other capillary electrophoresis techniques, CGE provides faster separations with higher resolution than slab gel versions (3). In high electric fields, nucleic acids become roughly oriented along the field direction and move at a velocity which is approximately size-independent (5-7). Changing the field direction disrupts the alignment and recovers size-dependent mobility. Because the time scale of nucleic acid motions is size-dependent, different field change intervals are necessary to optimize a separation for a given size range. These pulsed-field techniques have been most extensively developed for fragments larger than 10 kbp (thousands of base pairs) (8).
At the fields used in CGE (>lo0 V/cm) even short fragments align in gels, and resolution can suffer. Heiger, Cohen, and Karger have recently demonstrated that pulsed field operation improves the resolution of short restriction fragments in linear polyacrylamide-filled capillaries (4). For resolving the 4363- and 7253-bp fragments in 6% linear polyacrylamide, best results were obtained with 50% duty cycle and a frequency of about 100 Hz. They were unable to achieve resolution in this size range in conventional cross-linked gels. In the present communication, we report on the use of analyte velocity modulation capillary electrophoresis (9,lO) as a form of pulsed-field CGE. Sinusoidal changes in field strength and direction replace the customary step changes of pulsed-field electrophoresis. Analyte velocity modulation was developed to improve the performance of capillary zone electrophoresis (CZE) detectors that are excess noise limited. In CGE, an ac electric field of the proper frequency superimposed on the driving dc field would be expected to shorten nucleic acid fragment transit times and improve resolution in gel-filled capillaries. Although analyte velocity modulation does not change migration times or band shapes in CZE (9), it does increase the heat dissipation in an electrophoresis capillary, as a simple argument shows. With a superimposed ac field, the RMS voltage in the capillary is Vdc(l vdc/2vac)2)'/2,where Vd, and V,, are the dc and peak ac voltages, respectively. Thus, the RMS power is proportional to Vd:(1 + (vac/2vdc)2). Under the usual modulation conditions, V,, I0.5Vdc,and the excess power never exceeds 12.5%. Even when v,, = Vd,, the
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excess power is increased by 50%
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EXPERIMENTAL SECTION Electrophoresis was performed in 75 pm i.d. quartz capillaries (Polymicro Technologies, TSP/075/375). The total capillary length was 25 cm, with an entrance to detector length of 20 cm. The capillaries were filled with 3.5% T, 3.3% C polyacrylamide gel (Sigma), using the protocol of Paulus, Gassmann, and Field (11). Urea was not used in our gel preparations. The samples were 4X 174 RF DNA HaeIII fragments (Bethesda Research Laboratories). Separations were performed in 1X (90mM Tris) TBE buffer (Sigma) containing 0.5 rg/mL ethidium bromide. Electrophoretic separations were performed in the analyte velocity modulation apparatus previously described (9)using 180 V/cm dc and 0-120'70 depth of modulation (ratio of ac to dc voltage). A dc high voltage was applied between the capillary entrance and ground. A bucking voltage was applied between the capillary exit end and ground by an 88:l step-up transformer driven by a signal generator and power operational amplifier (Apex PA85). Detection of the ethidium bromide complexed nucleic acids was by laser-induced fluorescence,using 325-nm He-Cd laser illumination and observation at 580 nm with a sharp-cut filter (Wratten gelatin filter No. 23A) and a photomultiplier tube. The dc signal was amplified, low-pass-filtered,(
