Method for sequencing reaction cleanup by constant differential pressure ultrafiltration

• US 6,645,722 B2
• Filed: 10/19/2001
• Issued: 11/11/2003
• 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:
  US10/040,037
• Publication Number:
  US 20020102593A1
• Time in Patent Office: 753 Days
• Field of Search: 536/23.1, 435/91.1, 435/6, 210/650
• US Class Current: 435/6.12
• 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;

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; and

recovering said sequencing reaction product substantially free of said contaminants by one or more steps selected from the group consisting of pipetting, diffusion, agitation and electrokinetic transport.

2. The method of claim 1, wherein said recovery is by electrokinetic transport.

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 2, further comprising 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 comprises adding one or more solvents selected from the group consisting of formamide, EDTA and water, to said sequencing reaction.

6. The method of claim 2, 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.

7. 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 comprising one or more single nucleotide polymorphisms;

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

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

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.

8. The method of claim 7, further comprising recovering said sequencing reaction product substantially free of said contaminants by one or more steps selected from the group consisting of pipetting, diffusion, agitation and electrokinetic transport.

9. The method of claim 7, wherein said ultrafiltration membrane has a molecular weight cutoff between about 3,000 and 15,000 Daltons.

10. The method of claim 7, further comprising suspending said quantity of sequencing reaction product in a first solvent.

11. The method of claim 10, wherein said step of suspending said quantity of sequencing reaction product in a solvent comprises adding one or more solvents selected from the group consisting of formamide, EDTA and water, to said sequencing reaction.