Rapid separation and purification of oligonucleotides by high-performance capillary gel electrophoresis.

Abstract

Picomole amounts of oligodeoxynucleotides [polydeoxyadenylic acids, (dA)4&..s] were baseline resolved and analyzed in <8 min by high-performance capillary electrophoresis with polyacrylamide gels. In addition, fast analysis of a crude 70-mer oligodeoxynucleotide and a slab gel-purified 99-mer oligodeoxynucleotide was accomplished, demonstrating the ability of high-performance capillary electrophoresis to characterize rapidly synthesized oligonucleotides. Besides analytical separations, 800 ng of a primer (20-mer) was isolated in <20 min. The purified species was collected in water and subsequently used as a probe in a standard dot-blot analysis. The use of high-performance capillary electrophoresis for the analysis and purification of a variety of biopolymers is simple, rapid, and has the potential for automation. (7), was used at a wavelength of 260 nm. The tubing and the detector were cooled with a thermostated air bath. The power supply outlets were connected to platinum electrodes, 5, immersed in buffer reservoirs, 6 (for analytical runs), or in a microcentrifuge vial, 7 (for collection). An analog/digital interface, 8 (Nelson, Cupertino, CA), attached to a recorder, 9, and IBM PC/XT, 10, were used to record the results and process the data.Materials. The oligodeoxynucleotide (5' GCCACGTCCA-GATTTATCAG 3') in Fig. 4 was synthesized on a model 380A DNA synthesizer (Applied Biosystems, Foster City, CA) using the silica-based solid-phase method (8) and the proton-activated nucleoside phosphoramidate method with methoxyphosphoramidates (9). The side-chain protecting groups were liberated by treatment with ammonium hydroxide at 550C for 6 hr. After this step, the oligodeoxynucleotide was recovered by lyophilization and dissolved in water.Polydeoxyadenylic acid mixture (dA)40-60 was purchased from Pharmacia. The crude 70-mer and slab gel-purified 99-mer oligodeoxynucleotides were the gift of N. Bischoffer (Genentech, South San Francisco, CA Procedure. A modified version (10) of the polyacrylamide gel capillary column described in ref. 5 was filled with polyacrylamide gel (T = 7.5%, C = 3.3%) (11) in 7 M urea/0.1 mM Tris/0.25 mM borate, pH 8.3. The field applied to the gel was 400 V/cm. To remove impurities from the acrylamide gel, the capillary gel column was preelectrolyzed at 6 kV for 30-60 min. During electrophoresis, the capillary was maintained at room temperature. The samples were electrophoretically injected into the column by dipping the cathodic end of the capillary into an aqueous solution of the sample and applying a field of 400 V/cm for =10 sec.Abbreviations: HPCE, high-performance capillary electrophoresis. ITo whom reprint requests should be addressed. 9660The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Luteal phase serum cell-free DNA as a marker of failed pregnancy after assisted reproductive technology

“…Bypassing the PCR step conferred the advantage of directly evaluating the cell-free DNA derived from the entire genome without variations due to artefacts and inefficiencies in phenol-chloroform extraction and PCR amplification steps, and the limited primer targeted gene regions such as the K-ras oncogene (25)(26). However, the standard capillary electrophoresis assay possesses certain advantages such as continuous flow through analysis and direct nucleic acids detection set up on the capillary tube (20)(21)(22). The modified assay was based on two different techniques that optimized the volume-to-surface area ratio to focus the cell-free DNA on to a small area and the use of next-generation sensitive fluorochromes such as the SYBR Green stain.…”

“…The objective of this study was to determine and compare the relative concentration of cell-free DNA in the sera of nonpregnant versus pregnant patients after ART treatment. The capillary electrophoresis (CE) method (20)(21)(22) was modified to provide an inexpensive and rapid assay for cell-free DNA based on items commonly available in the ART laboratory.…”

Influence of the electrokinetic injection conditions on the separation of DNA fragments in capillary electrophoresis

“…Traditionally, DNA has been analyzed almost exclusively by slab-gel electrophoresis (SGE) and this is still an important tool in biochemistry and molecular biology laboratories all over the world. However, since its earlier stages CE has shown impressive advantages over SGE for the separation of DNA restriction fragments using either gel-filled capillaries 2 or polymeric solutions 3 and it is becoming widely accepted. Analysis and separation of DNA fragments has been performed by CE in a variety of ways.…”

“…The fraction of the total electric current carried by each ionic species in solution during the electrokinetic process can be expressed by the transference number (T DNA ) 27 which is given by the equation (2): (2) where C, Z and µ are, respectively, the concentration, the charge and the free solution mobility for DNA and for any ion "i". This equation shows that the injection process should be biased towards faster migrating small ions and shorter DNA fragments (it is necessary to take into account also that DNA samples may contain chloride ions, deoxy and dideoxynucleotides, phosphates, template DNA, primer excess, and other anionic species from buffers).…”

DNA Sequencing by Capillary Electrophoresis Using Short Oligonucleotide Primer Libraries

“…Furthermore, the throughput using single capillary instruments can be potentially increased through multiple capillary array instruments (15,24,28). Cross-linked, gelfilled capillaries have been used to obtain size-dependent separation of DNA sequencing fragments (3,7,11). Although extended sequences have been obtained with such matrices (1), these materials can be disadvantageous in CE because of potentially poor gel stability.…”