WO1994029005A1 - Agarose-plugged electroeluter - Google Patents

Abstract

The isolation of nucleic acids is a basic and critical step in most of the molecular biological studies. The nucleic acids are usually fractionated by size on a gel matrix by electrophoresis and they are to be purified from the gel matrix for further manipulation and analysis. The electroelution is a method of drawing nucleic acids out of the gel matrix into solution by electricity and a new design of electroeluter is presented here. A cylinder filled with a material that can hold nucleic acids stably is plugged with agarose at both ends. Sample gel slices are placed merged with the top agarose plug on cathode side. The nucleic acid is pulled out of the gel slice and into the cylinder and held by the trap material. When the elution is over, the top-plug is removed and the trap is collected together with the nucleic acid. The latter is then recovered by precipitation with alcohol.

Description

Agarose-Plugged Electroeluter

Technical Field

Molecular Biology and Genetic Engineering

Background Art

Nucleic acids are usually separated by electrophoresis on agarose or acrylamide gels. Purification of separated nucleic acid from the gel matrix is an obligatory and critical step in most of the molecular biological research. Diverse methods have been developed to recover the fractionated nucleic acids (1, 2). A common approach is the electroelution of nucleic acids out of the gel slice into the solution. Samples can be eluted into a dialysis bag, to a high-salt layer maintained in a V-channel using an apparatus commercially available (Electroeluter, IBI, USA) or to a DEAE-ion- exchange paper embedded in the trough just ahead of the target band. The bound DNA is later recovered by alcohol-precipitation. Another approach is the removal of gel matrix. Specially prepared agarose with low-gelling temperature (SeaPlaque, FMC,

USA) is popular because the gel could be liquefied at 65°C without denaturing the

DNA. Ordinary agarose could also be solubilized at room temperature in the presence of potassium idodide and recovered by adsorption to glass powder (GeneClean, BIOlOl, USA). Enzymatic digestion of agarose is also possible (β-Agarase, New

Eangland Biolab, USA).

References:

1. D. Moore, J. Chory, and R. K. Ribaudo (1994) Isolation and purification of large DNA restriction fragments from agarose gels, in Current Protocols in Molecular

Biology (F. M. Ansubel, et al., eds.), Supp. 26. John Wiley & Sons, Inc., New York.

2. J. Chory and A. S. Baldwin, Jr. (1993) Resolution and recovery of small DNA fragments, in Current Protocols in Molecular Biology (F. M. Ansubel, et al., eds.), Supp. 25. John Wiley & Sons, Inc., New York. Disclosure of Invention

We are presenting a new way of electroelution in which the material such as salt or ion-exchange resin trapping nucleic acids is contained within a cylinder plugged with agarose at both ends. A gel slice carrying nucleic acid is placed on top of the agarose plug of cathode side. The electric current pulls the nucleic acid into the cylinder where it stops migration due to the trap. At the end of the run, top-agarose plug is removed and the trap with the nucleic acid is collected. The nucleic acid is then recovered by alcohol-precipitation.

Brief Description of Drawings

A model of agarose-plugged electroeluter and details of the eluter trap The agarose-plugged electroeluter is composed of three major parts; upper buffer reservoir, trap part, and lower reservoir. A platinum wire (1, 2) is placed on each reservoir. The overall dimension is 12 cm (width) x 8 cm (depth) x 15 cm (height) and is made of acryl.

The trap part(3) is permanently attached to the upper reservoir(4). Half of the trap is protruding the bottom of the upper reservoir to be immersed in the buffer of the lower reservoir(5). The two reservoirs are connected solely by the narrow channels(6) in the trap part(3). The triangle-shaped upper part of the trap is the gel-receptacle(7). It leads to the tiered narrow channels (8, 9) below. To construct traps, the lower oriface(lθ) is plugged with 1% agarose at the beginning, and nucleic-acid-trapping material fills the cylinder. We recommend 9 M ammonium acetate or DEAE-cellulose resin for this trap. Then the upper oriface is plugged with melt agarose simultaneously with the placement of the sample gel slice to get embedded in the plug.

The tiered structure of the channel is to prevent the slippage of the agarose plug. The slope(14) of the bottom surface of the trap part is to prevent the accumulation of air bubbles.

When the elution is over, the buffer in the upper reservoir is drained though the drain port(15). The gel slice and upper agarose plug are removed and the trap layer is collected with a pasteur pipet. Best Mode for Carrying out the Invention

The apparatus can be build based on the above design and distributred commercially to scientists and engineers involved in life science.

Industrial Applicability

The apparatus should be manufactured in a factory with automatic processing. The apparatus is an analytical device and intended for use in indivisual laboratories. It is not for mass purification or isolation of biological material.

Claims

Claims

1. The agarose-plugged cylinder harnessing trap material.

2. Y-shaped eluter trap part with tiered cylinders as the implementation of the above (1).