Processes for the preparation and separation of macromolecules

• US 5,286,434 A
• Filed: 10/29/1991
• Issued: 02/15/1994
• Est. Priority Date: 04/24/1989
• Status: Expired due to Fees

• Current Assignee: Xerox Corporation (Xerox Holdings Corp.)
• Original Assignee: Xerox Corporation (Xerox Holdings Corp.)
• Inventors: Noolandi, Jaan, Slater, Gary, Turmel, Chantal
• Primary Examiner(s): Tung, T.
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US07/783,967
• Time in Patent Office: 840 Days
• Field of Search: 204/299 R, 204/182.8, 204/182.1
• US Class Current: 204/457
• 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 27/44773 Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

Abstract

A process for the electrophoretic separation of charged macromolecules includes applying to the macromolecules a periodic sequence of pulses. Each period comprises a multiplicity of electric field pulses of negative and positive polarities. The negative polarity pulses are applied for a longer total time duration than the positive polarity pulses within each period. The average intensity of the negative polarity pulses is less than the average intensity of the positive polarity pulses.

Claims

1. A process for the separation of a mixture of DNA fragments or molecules comprising:

adding to an electrophoretic device an agarose gel which contains a solution mixture or an agarose plug containing DNA fragments of different lengths;

energizing the device, thereby creating a sequence of uniform electric field pulses along a single dimension therein, said sequence of field pulses alternating between positive polarity pulses and negative polarity pulses having less voltage than the positive polarity pulses; and

applying in the device selected positive polarity pulses and negative polarity pulses with intensities and duration corresponding to the size of fragments to be separated, wherein the negative polarity pulses are of a longer duration than the positive polarity field pulses;

the fragments to be separated possess between about 2,000 and 6,000,000 base pairs;

a concentration of agarose in the gel is between about 0.2 percent and about 2 percent;

the positive polarity pulse have a strength of between about +0.50 and about 4.0 volts/centimeter, and the electric current of the positive polarity pulses is between about 4 and about 80 milliamperes;

the positive polarity pulses are applied for about 1 second to about 1,000 seconds;

the negative polarity pulses are applied for about 1 to about 2,000 seconds; and

the negative polarity pulses have a duration about 1.4 times longer than the primary field pulses.

2. A process in accordance with claim 1, wherein the voltage for the positive polarity pulses is about 82 volts, the voltage for the negative polarity pulses is about negative 42 volts, the distance between the electrodes contained in the electrophoresis device is about 31 centimeters, the positive polarity pulse durations and the negative polarity pulse durations are selected from the group consisting of about 2 and 2.8 seconds;

5 and 7 seconds;

10 and 14 seconds;

30 and 42 seconds;

50 and 70 seconds; and

160 and 224 seconds.