Rapid and efficient capture of DNA from sample without using cell lysing reagent

• US 7,262,006 B1
• Filed: 05/01/2000
• Issued: 08/28/2007
• Est. Priority Date: 05/01/2000
• Status: Expired due to Fees

• Current Assignee: Ortho-Clinical Diagnostics Incorporated (The Carlyle Group LP (Corporate Private Equity))
• Original Assignee: Ortho-Clinical Diagnostics Incorporated (The Carlyle Group LP (Corporate Private Equity))
• Inventors: Sun, Jianbo, Belly, Robert T.
• Primary Examiner(s): Kim; Young J.
• Application Number:
  US10/019,514
• Time in Patent Office: 2,675 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1
• US Class Current: 435/6.1
• 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 2527/125 Specific component of sample, medium or buffer

Abstract

Nucleic acids can be made available for amplification or other treatment after admixture of a sample with specific weakly basic polymers to form a precipitate with the nucleic acids at acidic pH. After removing non-precipitated materials, the pH is then made basic, thereby releasing the nucleic acids from the polymer. This method for preparing specimen samples is simple and quite rapid, and the released nucleic acids can be further treated in hybridization assays or amplification procedures. No surfactant or other cell lysing reagents are employed. The weakly basic polymers are water-soluble and cationic at acidic pH, but neutral in charge at basic pH.

Claims

1. A method for isolating a free circulating, extracellular nucleic acid from a mammalian species sample not previously treated with a cell lysing reagent, wherein said method comprises the steps of:

A) admixed in buffer at a pH of less than 7, contacting a sample suspected of containing a nucleic acid with a water-soluble, weakly basic polymer derived by addition polymerization of;

1) from about 15 to 100 weight percent of a water-soluble, weakly basic ethylenically unsaturated polymerizable monomer having at least one group which can be protonated at acidic pH and which is selected from the group consisting of aminoalkyl, imidazolyl, isoxazolyl, pyridyl, piperidyl, piperazinyl, pyrazolyl, triazolyl, tetrazolyl, oxadiazolyl, pryidazinyl, pyrimidyl, pyrazinyl, quinolinyl and quinazolinyl,

2) from 0 to about 35 weight percent of a nonionic, hydrophilic ethylenically unsaturated polymerizable monomer, and

3) from 0 to about 85 weight percent of a nonionic, hydrophobic ethylenically unsaturated polymerizable monomer in an amount sufficient to form a water-insoluble precipitate of said weakly basic polymer with all nucleic acids present in said lysate,

B) separating said water-insoluble precipitate from said sample, and

C) contacting said precipitate with a base to raise the buffer pH to greater than 7, and thereby releasing said free circulating, extracellular nucleic acids from said weakly basic polymer,

said weakly basic polymer derived by addition polymerization of one or more ethylenically unsaturated polymerizable monomers having an amine group which can be protonated at acidic pH.

2. The method of claim 1 further comprising the step:

D) adjusting the pH of said solution containing said released nucleic acids to from about 6 to about 9.

3. The method of claim 1 wherein said base is sodium hydroxide, potassium hydroxide, ammonium hydroxide, lithium hydroxide, sodium carbonate, sodium bicarbonate, a tertiary amine or tris(hydroxymethyl)-aminomethane.

4. The method of claim 1 wherein said weakly basic polymer is used in step A) in an amount of from about 0.01 to about 0.5 weight %.

5. The method of claim 1 wherein a weak base is used in step C), accompanied by heating said water-insoluble precipitate at from about 50°

to about 125°

C.

6. The method of claim 1 wherein a strong base is used in step C) without heating said water-insoluble precipitate.