Method and apparatus for compression of DNA samples for DNA sequencing

• US 6,475,362 B1
• Filed: 12/03/1999
• Issued: 11/05/2002
• Est. Priority Date: 12/03/1998
• Status: Expired due to Term

• Current Assignee: The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Gorfinkel, Vera, Gouzman, Mikhail, Serge, Luryi
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US09/454,094
• Time in Patent Office: 1,068 Days
• Field of Search: 204/451, 204/601, 204/453, 204/455, 204/604, 204/605
• US Class Current: 204/451
• CPC Class Codes: G01N 27/44743 Introducing samples

Abstract

A novel method of spatial compression of a DNA sample inside the capillary for the gel capillary electrophoresis and an article for operating the method are disclosed. In this method, after the electrokinetic injection, the sample is compressed using the reverse electric field. A special DNA barrier material is used to contain the sample in the capillary. We expect that this Electro Static Compression (ESC) can increase the DNA concentration in the capillary by orders of magnitude. In the proposed method and article the injection and compression of the DNA sample are followed by subsequent electrophoretic separation in any kind of sequencing container (for instance glass capillary). The use of the ESC method will allow the increase of the length and the quality of the read, as well as reduction of the DNA consumption. ESC method will also permit the use of low power illumination sources including miniature and inexpensive light emitting diodes (LED) instead of the Ar-ion laser for exciting fluorescent labels in 4-color DNA sequencing.

Claims

1. A method of spatial compression of a DNA sample inside a capillary for gel capillary electrophoresis comprising the steps of:

providing a capillary having an inlet end and an outlet end;

providing a first electrode adjacent said capillary inlet end and a second electrode adjacent said capillary outlet end;

electrophoretically injecting DNA sample into said inlet end of said capillary by applying a voltage across said first electrode and said second electrode;

contacting the inlet end of the capillary with DNA barrier material;

compressing the DNA sample against the DNA barrier material by applying a voltage having a polarity reversed to the electrokinetic injection voltage across said first electrode and said second electrode; and

electrophoretically separating the compressed DNA sample by applying a voltage having a polarity reversed to the compression voltage.

2. The method of claim 1, wherein the compressed sample occupies about less than 40 μ

m.

3. The method of claim 1, wherein the DNA barrier comprises a high-density PAA gel including about 30% T and 5% C.

4. The method of claim 3, further comprising the step of limiting the compression by a space charge at an interface of the aluminum foil, wherein the DNA sample does not adhere to the aluminum foil.

5. The method of claim 1, wherein the DNA barrier comprises an aluminum foil disposed at a lower portion of the capillary.

6. The method of claim 1, wherein an electrophoretically separate sample can be viewed under a single photon detection module.

7. The method of claim 6, wherein the single photon detection module comprises a dynamic range of about 23 bits.

8. The method of claim 6, wherein down to about 100 photons can be used for identification of a fluorescent dye in the DNA sample.

9. The method of claim 6, wherein the single photon module further comprises a four-color beam wherein each color is modulated at a frequency range of about between 100 and 300 Hz.

10. The method of claim 1 wherein the step of electrokinetically injecting the DNA sample is performed at a voltage of about 16 kV and current of about 10 μ

A for about 180 seconds.

11. The method of claim 1 wherein the step of electrokinetically injecting the DNA a sample is performed at a voltage less than about 20 kV and current less than about 10 μ

A.