METHODS AND DEVICES FOR NUCLEIC ACID PURIFICATION

• US 20120252115A1
• Filed: 03/29/2012
• Published: 10/04/2012
• Est. Priority Date: 03/29/2011
• Status: Active Grant

• Current Assignee: PhyNexus, Inc.
• Original Assignee: PhyNexus, Inc.
• Inventors: Suh, Chris, Hoang, Lee, Gjerde, Douglas T.
• Granted Patent: US 8,921,539 B2
• Time in Patent Office: Days
• US Class Current: 435/320.1
• CPC Class Codes: B01D 15/322 Normal bonded phase
• CPC Class Codes: B01D 15/361 Ion-exchange
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 3/0275 Interchangeable or disposable dispensing tips
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• 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: G01N 2035/1039 Micropipettes, e.g. microcapillary tubes

Abstract

The invention provides pipette tip columns and automated methods for the purification of nucleic acids such as plasmids from unclarified cell lysates containing cell debris as well as methods for making and using such columns. The columns typically include a bed of medium positioned in the pipette tip column, above a bottom frit and with an optional top frit.

Claims

1. An automated method for purifying plasmid DNA with a pipette tip column, the method comprising:

i) providing a plurality of unclarified lysates comprised of plasmid DNA, liquid and particulates;

ii) providing a plurality of pipette tip columns, wherein the pipette tip columns are arranges in a 9 mm center-to-center format, and wherein the pipette tip columns contain a solid phase having an affinity for plasmid DNA;

iii) capturing the unclarified lysates on the pipette tip columns;

iv) passing a wash solution through the pipette tip columns, wherein the wash solution is comprised of an organic solvent;

v) optionally, repeating step (iv);

vi) using vacuum to pass air through the pipette tip columns for at least 30 seconds; and

vii) eluting the plasmids by passing an aqueous solution through the pipette tip columns, wherein the organic solvent content of the eluted plasmid is 5% or less.

2. The method of claim 1, wherein one or more of the sample solution, the wash solution and the elution solution is passed through the pipette tip columns by gravity flow.

3. The method of claim 1, wherein one or more of the sample solution, the wash solution and the elution solution is passed through the pipette tip columns using vacuum.

4. The method of claim 1, wherein one or more of the sample solution, the wash solution and the elution solution is passed through the columns by aspirating and expelling the solution through the open lower end of the pipette tip columns.

5. The method of claim 4, wherein the eluted plasmid has an endotoxin content of less than 0.01 endotoxin units per microgram of DNA.

6. An automated method for capturing plasmid DNA from an unclarified lysate with a pipette tip column, the method comprising:

i) providing a plurality of unclarified lysates having a sample volume, wherein each unclarified lysate is comprised of plasmid DNA, liquid and particulates;

ii) providing a plurality of pipette tip columns arranged in a 9 mm center-to-center format;

iii) submerging the open lower end of the pipette tip columns in the unclarified lysates;

iv) aspirating and expelling a portion of the unclarified lysates through the open lower end of the pipette tip columns, wherein the portion of each unclarified lysate aspirated is less than 80% of the sample volume; and

v) optionally, repeating step (iv).

7. The method of claim 6, wherein the portion of the unclarified lysate is less than 60% of the sample volume.

8. The method of claim 7, wherein the method is performed on at least 2 and at most 96 pipette tip columns simultaneously.