RAPID AND EFFICIENT CAPTURE OF DNA FROM SAMPLE WITHOUT USING CELL LYSING REAGENT

• US 20070243542A1
• Filed: 12/20/2006
• Published: 10/18/2007
• Est. Priority Date: 05/04/1999
• Status: Active Grant

• Current Assignee: Jianbo Sun, Robert Belly
• Original Assignee: Jianbo Sun, Robert Belly
• Inventors: Sun, Jianbo, Belly, Robert
• Granted Patent: US 7,615,346 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• 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 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

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. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. A method for the amplification and detection of a target nucleic acid without the use of a cell lysing reagent, comprising:

I) providing a sample suspected of containing a target nucleic acid,

II) subjecting said sample containing the target nucleic acid to the steps of;

A) at a pH of less than 7, contacting said target nucleic acid with a water-soluble, weakly basic polymer comprised of recurring units 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, pyridazinyl, 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 sample, including said target nucleic acid,

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

C) contacting said precipitate with a base to raise the solution pH to greater than 7, and thereby releasing said nucleic acids, including said target nucleic acid, from said weakly basic polymer,

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

III) without further adjustment of pH, amplifying said released target nucleic acid, and

IV) detecting said amplified target nucleic acid.

8. The method of claim 7 wherein said weakly basic polymer is water-insoluble at basic pH, and said method further comprises the step of removing said water-insoluble polymer after release of said target nucleic acid therefrom and prior to amplification thereof.

9. The method of claim 7 wherein the target nucleic acid is a K-ras sequence.

10. A kit for detection of K-ras mutation in a biological sample, said kit comprising a diagnostic K-ras primer selected from the group consisting of:

(a) TGAATATAAA CTTGTGGTAC CTGGACC T (5K15S) <

SEQ ID;

NO 1>

,

(b) ATATAAACTT GTGGTACTTC CAGCTGGT (5K37) <

SEQ ID;

NO 2>

,

(c) GAATTAGCTG TATCGTCAAG GCACTC (3K42) <

SEQ ID;

NO 3>

,

(d) TCAGCAAAGA CAAGACAGGT A (5BK5) <

SEQ ID;

NO 4>

,

(e) TATAGATGGT GAAACCTGTT TGTTGG (5N12A) <

SEQ ID;

NO 5>

,

(f) CTTGCTATTA TTGATGGCAA CCACACAGA (3N13A) <

SEQ ID;

NO 6>

(g) any combination of the foregoing.

11. The oligonucleotide TGAATATAAA CTTGTGGTAC CTGGAGC T <

SEQ ID;

NO 1>

.

12. The oligonucleotide ATATAAACTT GTGGTAGTTC CAGCTGGT <

SEQ ID;

NO 2>

.

13. The oligonucleotide GAATTAGCTG TATCGTCAAG GCACTC <

SEQ ID;

NO 3>

.

14. The oligonucleotide TCAGCAAAGA CAAGACAGGT A <

SEQ ID;

NO 4>

.

15. The oligonucleotide TATAGATGGT GAAACCTGTT TGTTGG <

SEQ ID;

NO 5>

.

16. The oligonucleotide CTTGCTATTA TTGATGGCAA CCACACAGA <

SEQ ID;

NO 6>

.

17. A K-ras diagnostic primer comprising the oligonucleotide TGAATATAAA CTTGTGGTAC CTGGAGC T <

SEQ ID;

NO 1>

.

18. A K-ras diagnostic primer comprising the oligonucleotide ATATAAACTT GTGGTAGTTC CAGCTGGT <

SEQ ID;

NO 2>

,

19. A K-ras diagnostic primer comprising the oligonucleotide GAATTAGCTG TATCGTCAAG GCACTC <

SEQ ID;

NO 3>

.

20. A K-ras diagnostic primer comprising the oligonucleotide TCAGCAAAGA CAAGACAGGT A <

SEQ ID;

NO 4>

.

21. A K-ras diagnostic primer comprising the oligonucleotide TATAGATGGT GAAACCTGTT TGTTGG <

SEQ ID;

NO 5>

.

22. A K-ras diagnostic primer comprising the oligonucleotide CTTGCTATTA TTGATGGCAA CCACACAGA <

SEQ ID;

NO 6>

.