Method for purification and transfer to separation/detection systems of DNA sequencing samples and plates used therefor

• US 5,856,100 A
• Filed: 12/06/1996
• Issued: 01/05/1999
• Est. Priority Date: 12/08/1995
• Status: Expired due to Term

• Current Assignee: The Institute of Physical and Chemical Research
• Original Assignee: The Institute of Physical and Chemical Research
• Inventors: Hayashizaki, Yoshihide
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): SHOEMAKER, DEBRA GUNNERSEN
• Application Number:
  US08/764,107
• Time in Patent Office: 760 Days
• Field of Search: 435/6, 435/5, 435/91.2, 435/284, 435/809, 435/305.3, 435/305.2, 435/288.5, 435/288.6, 204/455, 204/451, 204/601, 536/25.4
• US Class Current: 435/6.11
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00367 capillary
• CPC Class Codes: B01J 2219/00416 Vacuum
• CPC Class Codes: B01J 2219/00423 using filtration, e.g. through porous frits
• CPC Class Codes: B01L 3/50255 Multi-well filtration
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 27/44743 Introducing samples

Abstract

Disclosed are: a part for assembling reaction vessels comprising a base plate with holes passing through said plate in the direction of thickness; a method for preparation of reaction vessels containing a reaction solution characterized in that the part is immersed in a reaction solution to introduce the reaction solution into the holes and then one of apertures of each hole is sealed with a membrane to complete said reaction vessels; and a plate comprises a base plate with holes passing through the plate in the direction of thickness and a membrane for sealing one of the apertures of each hole. The plate is used for purification of DNA sequencing samples using a pressure difference or an electrical potential, and transferring DNA sequencing samples to a separation/detection system. By the means or the method of the invention, reaction solutions can be easily introduced into a large number of wells in a short time, unreacted labeling substances and the like can be easily removed in a short time from a plurality of DNA sequencing samples, and a large number of DNA sequencing samples can be easily purified and/or transferred to electrophoresis capillaries in a short time.

Claims

1. A method for preparation of reaction vessels containing a reaction solution comprising immersing a part comprising

a base plate with holes passing completely through said plate such that each hole has two apertures, one on each side of said base plate, and wherein

each of said holes is suitable for containing reaction solutions when one of the apertures of said hole is sealed,

in a reaction solution introduce the reaction solution into the holes, and then sealing one of the apertures of each hole with a membrane, thus forming reaction vessels containing said reaction solution.

2. The method of claim 1 wherein the reaction solution is a buffer solution containing an enzyme.

3. A method for removal of unreacted low molecular weight compounds contained in DNA sequencing samples comprising introducing said DNA sequencing samples into the holes of a plate, said plate comprising a base plate with holes passing completely through said plate such that each hole has two apertures, one on each side of said base plate, and an ultrafiltration membrane sealing one of the apertures of each hole wherein said membrane comprises an electrically conductive material or a liquid-permeable material, creating a pressure difference between the opening of each hole and the outside of the sealing membrane such that the pressure is relatively lower on the membrane side, thereby transferring said unreacted low molecular weight compounds from the DNA sequencing samples through said membrane.

4. The method of claim 3 wherein the unreacted low molecular weight compounds are unreacted fluorescence labeling compounds.

5. The method of claim 3 wherein said DNA sequencing samples are products of DNA sequencing reactions conducted in reaction solutions contained by the plate.

6. The method of claim 3 wherein said DNA sequencing samples are products of DNA sequencing reactions conducted in a micro-titer plate.

7. A method for removal of unreacted low molecular weight compounds contained in DNA sequencing samples, comprising introducing said DNA sequencing samples into the holes of a plate, said plate comprising a base plate with holes passing completely through said plate such that each hole has two apertures, one on each side of said base plate, and an ultrafiltration membrane sealing one of the apertures of each hole wherein said membrane comprises an electrically conductive material or a liquid-permeable material, and applying a potential between said DNA sequencing samples and the outside of the sealing membrane such that said unreacted low molecular weight compounds in the DNA sequencing samples are transferred through said membrane.

8. The method of claim 7, further comprising inserting one terminal of electrophoretic capillaries into the DNA sequencing samples, contacting the other terminal of said capillaries with an electrolyte contacting an electrode (1);

contacting the sealing membrane with an electrolyte with which an electrode (2) is contacted; and

applying a potential between the electrodes (1) and (2).

9. The method of claim 7, further comprising inserting electrodes into the DNA sequencing samples contacting an electrode with the electrolyte;

and applying a potential between the electrodes inserted in the DNA sequencing samples and the electrode contacted with the electrolyte.

10. The method of claim 7 wherein the unreacted low molecular weight compounds are unreacted fluorescence labeling compounds.

11. The method of claim 7 wherein said DNA sequencing samples are products of DNA sequencing reactions conducted in reaction solutions contained by the plate.

12. The method of claim 7 wherein said DNA sequencing samples are products of DNA sequencing reactions conducted in the wells of a micro-titer plate.

13. A method for purification and transfer to a separation and/or detection system of DNA sequencing samples, which comprises removing unreacted low molecular weight compounds contained in the DNA sequencing samples and then transferring target substances contained in the DNA sequencing samples to electrophoretic capillaries for the separation and/or detection system, wherein said DNA sequencing samples are introduced into the holes of a plate, said plate comprising a base plate with holes passing completely through said plate such that each hole has two apertures, one on each side of said base plate, and an ultrafiltration membrane sealing one of the apertures of each hole wherein said membrane comprises an electrically conductive material or a liquid-permeable material, one terminal of an electrophoretic capillary is inserted into each of said DNA sequencing samples, a potential is applied between the outside of said membrane sealing the holes containing said DNA sequencing samples and the other terminal of each capillary of the electrophoretic capillaries such that unreacted low molecular weight compounds contained in said DNA sequencing samples are transferred through the membrane, and then a potential is applied between the outside of said membrane, and the other terminal of each electrophoretic capillary to transfer said target substances in the DNA sequencing samples into the electrophoretic capillaries.

14. A method for purification and transfer to a separation and/or detection system of DNA sequencing samples, which comprises removing unreacted low molecular weight compounds contained in the DNA sequencing samples and then transferring target substances contained in the DNA sequencing samples to electrophoretic capillaries for the separation and/or detection system, characterized in that said DNA sequencing samples are charged in the holes of a plate, said plate comprising a base plate with holes passing completely through said plate such that each hole has two apertures, one on each side of said base plate, and an ultrafiltration membrane sealing one of the apertures of each hole wherein said membrane comprises an electrically conductive material or a liquid-permeable material, a pressure difference is provided between the remaining apertures of the holes and the outside of the sealing membrane in such a manner that the membrane side should have negative pressure to transfer said unreacted low molecular weight compounds in the DNA sequencing samples to the outside of the samples through said membrane, then one of terminals of each capillary of electrophoretic capillaries are inserted into said DNA sequencing samples, and a potential is applied between the outside of said membrane sealing the holes charged with said DNA sequencing samples and the other terminal of each capillary of the electrophoretic capillaries to transfer target substances contained in said DNA sequencing samples to the electrophoretic capillaries.