Process and apparatus for purifying and concentrating DNA from crude mixtures containing DNA

• US 4,617,102 A
• Filed: 03/21/1985
• Issued: 10/14/1986
• Est. Priority Date: 03/21/1985
• Status: Expired due to Fees

• Current Assignee: Lifecodes Corporation (Laboratory Corporation Of America Holdings)
• Original Assignee: Lifecodes Corporation (Laboratory Corporation Of America Holdings)
• Inventors: Ford, John P., Tomblin, Graham J., Wexler, Karen B., Fischer, Stuart G.
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Boggs, Jr., B. J.
• Application Number:
  US06/714,376
• Time in Patent Office: 572 Days
• Field of Search: 204/299 R, 204/301, 204/182.1, 204/182.3, 204/182.6, 204/182.8
• US Class Current: 204/613
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• CPC Class Codes: G01N 27/447 using electrophoresis

Abstract

A process and apparatus for purifying and concentrating DNA, which has relatively high molecular weight, from a crude mixture containing DNA for example from human whole blood samples; the collected DNA is of usable volume and concentration, and is of such purity as to permit conventional restriction by a number of enzymes without the need for further purification. The apparatus consists essentially of an agarose gel disc, typically 5 mm. thick by 31 mm. in diameter, immersed in an electrophoresis buffer solution and supported between two 8 micrometer polycarbonate filters in an electric field. Further, the method involves the loading of a suitably treated sample such as blood lysate onto the top face of the agarose gel disc and then applying an electric field. On the constituent parts of the treated blood, the DNA molecules are the largest with regard to molecular weight. Consequently, their passage through the agarose gel disc, under the force of the electric field, is impeded. All other constituent parts of the treated blood pass relatively rapidly through the disc and are removed, being swept away by flow of the buffer solution. The DNA is then eluted and collected, in concentrated form by application of the electric field, whilst the normal flow of buffer solution between the bottom of the gel matrix and a collection chamber is prevented.

Claims

1. Apparatus for purifying and concentrating DNA from a crude mixture containing the DNA comprising:

a container providing a reservoir of electrophoresis buffer solution;

a gel matrix immersed in said electrophoresis buffer solution;

a collection chamber in said container;

means for applying an electric field to said gel matrix;

means for disposing said crude mixture in contact with the gel matrix such that the crude mixture is drawn into the gel matrix, responsive to said electric field;

means for selectively removing, due to their relatively low molecular weight, all the constituent molecules of the crude mixture, except for the DNA, from said gel matrix responsive to said electric field, including means for sweeping away said constituent molecules by flow of said electrophoresis buffer solution between the bottom of the gel matrix and the collection chamber; and

means for removing from the gel matrix, responsive to said electric field, the DNA still remaining therein such that the DNA is collected in said collection chamber, including means operative when removing the DNA for preventing said flow of said electrophoresis buffer solution, thereby to avoid sweeping away the DNA.

2. Apparatus as defined in claim 1, in which said crude mixture is human whole blood which has been treated.

3. Apparatus as defined in claim 2, in which said crude mixture is human whole blood which has been mixed with a lysis buffer.

4. Apparatus as defined in claim 1, further comprising an extraction assembly, and in which said means for applying an electric field includes a positive electrode and a negative electrode connected to spaced locations on said assembly.

5. Apparatus as defined in claim 4, in which said extraction assembly comprises a gel disc sub-assembly and a manifold sub-assembly, said positive electrode being connected to said gel disc sub-assembly and said negative electrode to said manifold sub-assembly.

6. Apparatus as defined in claim 5, in which said positive electrode is a point electrode located near the bottom of said extraction assembly.

7. Apparatus as defined in claim 6, further including means for preventing the accumulation of bubbles adjacent said positive, point electrode by pumping the buffer solution rapidly over said electrode.

8. Apparatus as defined in claim 5, in which said gel matrix is an agarose gel disc and two filters, and said manifold sub-assembly includes a top manifold, a bottom manifold, and a bottom block.

9. Apparatus as defined in claim 8, in which said bottom manifold is provided with an opening for said flow of buffer solution in sweeping away said constituent molecules.

10. An apparatus as defined in claim 1, in which said gel matrix has an upper face and a lower face, and further in which said means for disposing the test sample enables contacting said upper face of said gel matrix with said sample.

11. Apparatus as defined in claim 1, in which said means for selectively removing includes means for initially positioning said gel at a first level within said electrophoresis buffer solution.

12. Apparatus as defined in claim 11, in which said means for removing the DNA from the gel matrix includes means for positioning said gel matrix at a lower level than said first level.

13. Apparatus as defined in claim 12, in which said applied electric field has a first value when said gel matrix is positioned at said first level, and said electric field has a second value when said gel matrix is at said lower level.

14. Apparatus as defined in claim 13, including means for preventing the DNA from being swept away by the flow of electrophoresis buffer solution, while applying an electric field sufficient to remove the DNA from the gel, whereby the DNA is collected for further testing.