Method for sequencing reaction cleanup by constant pressure diffential ultrafiltration

• US 6,498,240 B1
• Filed: 07/24/2000
• Issued: 12/24/2002
• Est. Priority Date: 09/17/1999
• Status: Expired due to Term

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Original Assignee: Millipore Corporation
• Inventors: MacDonald, Constance, Gabriels, Joseph, Leonard, Jack T.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/624,174
• Time in Patent Office: 883 Days
• Field of Search: 435/91.1, 435/6, 200/650, 536/23.1
• US Class Current: 536/23.1
• CPC Class Codes: B01D 61/145 Ultrafiltration
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/109 Pressure
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/15 Gradients
• CPC Class Codes: C12Q 2547/107 Use of permeable barriers, e.g. waxes
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation

Abstract

A method for sequencing reaction cleanup adapted to remove contaminants from a sequencing reaction, comprising the steps of: providing a defined quantity of sequencing reaction product; providing at least one ultrafiltration membrane having at least one surface; transferring the suspended sequencing reaction product to the surface of the ultrafiltration membrane; and applying a first constant pressure differential to the ultrafiltration membrane at a force capable of producing the sequencing reaction product substantially free of the contaminants.

Claims

1. A method for sequencing reaction cleanup adapted to remove contaminants from a sequencing reaction, comprising the steps of,

providing a quantity of sequencing reaction product;

providing at least one ultrafiltration membrane having at least one surface;

transferring said suspended sequencing reaction product to said surface of said ultrafiltration membrane; and

applying a first constant pressure differential to said ultrafiltration membrane at a force capable of producing said sequencing reaction product substantially free of said contaminants, said force being between about 5 to 28 inches of mercury.

2. The method of claim 1, wherein said ultrafiltration membrane has a molecular weight cutoff between about 1000 and 30,000 Daltons.

3. The method of claim 2, wherein said ultrafiltration membrane has a molecular weight cutoff between about 3,000 and 15,000 Daltons.

4. The method of claim 1, further comprising the step of suspending said quantity of sequencing reaction product in a first solvent.

5. The method of claim 4, wherein said step of suspending said quantity of sequencing reaction product in a solvent comprising adding one or more solvents, selected from a group consisting of formamide, EDTA and water, to said sequencing reaction.

6. The method of claim 1, wherein said sequencing reaction product comprises one or more single nucleotide polymorphisms.

7. The method of claim 1, further comprising the steps of,

washing said sequencing reaction product substantially free of said contaminants with a second solvent; and

applying a second constant pressure differential to said ultrafiltration membrane.

8. The method of claim 7, wherein said second constant pressure differential is applied at a force between about 5 to 28 inches of mercury.

9. The method of claim 7, wherein said second solvent comprises one or more solvent selected from the group consisting of formamide, water and EDTA.

10. The method of claim 7, further comprising the steps of,

washing said sequencing reaction product substantially free of said contaminants with a third solvent;

and

applying a third constant pressure differential to said ultrafiltration membrane.

11. The method of claim 10, wherein said third solvent comprises one or more solvent selected from the group consisting of water, formamide and EDTA.

12. The method of claim 10, wherein said third constant pressure differential is applied at a force between about 5 and 28 inches of mercury.

13. The method of claim 1, further comprising the step of recovering said sequencing reaction product substantially free of said contaminants by pipetting off said surface of said membrane.

14. The method of claim 13, wherein a vacuum of about 5 inches of mercury is applied while pipetting off said surface of said membrane.

15. The method of claim 1, wherein said contaminants comprise salt and wherein said step of applying a first constant pressure differential to said ultrafiltration membrane comprises applying a force capable of producing said sequencing reaction product substantially free of salts.

16. The method of claim 1, wherein said contaminants comprise dye terminators and wherein said step of applying a first constant pressure differential to said ultrafiltration membrane comprises applying a force capable of producing said sequencing reaction product substantially free of dye terminators.

17. The method of claim 1, wherein said ultrafiltration membrane has an upstream and a downstream surface and wherein said suspended sequencing reaction product is transferred to said upstream surface.

18. The method of claim 17, further comprising the step of recovering said sequencing reaction product substantially free of said contaminants by pipetting off said upstream surface of said membrane.

19. The method of claim 1, further comprising the step of recovering said sequencing reaction product substantially free of said contaminants by one or more steps selected from a group consisting of pipetting, diffusion, agitation and electrokinetic transport.

20. The method of claim 19, wherein a vacuum of about 5 inches of mercury is applied during said step of recovering.

21. The method of claim 1, further comprising the step of washing said sequencing product with one or more salts capable of displacing any sodium ions from said product.

22. The method of claim 1, further comprising the step of resuspending said sequencing product, substantially free of one or more of said contaminants, in a matrix capable of crystallization.

23. The method of claim 1, further comprising the step of resuspending said sequencing product, substantially free of one or more of said contaminants, in water.

24. The method of claim 1, further comprising the step of resuspending said sequencing product, substantially free of one or more of said contaminants, in one or more volatile solvents.

25. A method for sequencing reaction cleanup adapted to remove contaminants from a sequencing reaction, comprising the steps of,

providing a quantity of sequencing reaction product;

suspending said quantity of sequencing reaction product in one or more solvents selected from a group consisting of formamide, EDTA and water;

providing at least one ultrafiltration membrane having at least one surface;

transferring said suspended sequencing reaction product to said surface of said ultrafiltration membrane; and

applying a first constant pressure differential to said ultrafiltration membrane at a force between about 5 to 28 inches of mercury to produce said sequencing reaction product substantially free of said contaminants.

26. The method of claim 25, wherein said sequencing reaction product comprises one or more single nucleotide polymorphisms.

27. The method of claim 25, further comprising the steps of,

washing said sequencing reaction product substantially free of said contaminants with a second solvent; and

applying a second constant pressure differential to said ultrafiltration membrane at a force between about 5 to 28 inches of mercury.

28. The method of claim 27, wherein said second solvent comprises one or more solvents selected from a group consisting of formamide, water and EDTA.

29. The method of claim 28, further comprising the steps of,

washing said sequencing reaction product substantially free of said contaminants with a third solvent;

and

applying a third constant pressure differential to said ultrafiltration membrane.

30. The method of claim 25, wherein said contaminants comprise one or more salts and one or more dye terminators and wherein said step of applying a first constant pressure differential to said ultrafiltration membrane comprises applying a force capable of producing said sequencing reaction product substantially free of said salts and said dye terminators.

31. The method of claim 25, further comprising the step of resuspending said sequencing product, substantially free of one or more of said contaminants, in a matrix capable of crystallization.

32. The method of claim 25, further comprising the step of resuspending said sequencing product, substantially free of one or more of said contaminants, in one or more volatile solvents.