WO2006083962B1

WO2006083962B1 - Selected compaction agents and methods and compositions for biotechnical separations

Info

Publication number: WO2006083962B1
Application number: PCT/US2006/003548
Authority: WO
Inventors: Richard Don Goodin; Iii Richard Coale Willson
Original assignee: Technology Licensing Co Llc; Richard Don Goodin; Iii Richard Coale Willson
Priority date: 2005-02-03
Filing date: 2006-02-01
Publication date: 2007-08-02
Also published as: EP1848824A4; WO2006083962A1; EP1848824A1

Abstract

Disclosed herein, is a method of isolating nucleic acid molecules from a sample, wherein isolation is achieved by adding a compaction agent. Also disclosed herein are methods of manipulating the isolated nucleic acid molecules.

Claims

AMENDED CLAIMS received by the International Bureau on 30 August 2006 (30.08.2006)

A. A method of preparing substantially purified DNA, without the use of nucleases or proteases, by adding an effective amount of a compaction agent selected from the group consisting of: quaternary ammonium polyamines, the compounds of FTGS. 1-3 and PTCs to a lysate to precipitate from said lysate, DNA having a content of RNA of less than 3% by weight.

2. The method of Claim 19 wherein the reaction comprises messengεr-RNA (mRNA) and DNA monomer nucleotides to produce cDNA under action of reverse transcriptase.

3. The method of Claim 2 wherein the cDNA is a copy of the mRNA, reverse-transcribed into

DNA form by a template-guided polymerization.

4. The method of Claim 19 wherein the reaction product is detected by methods comprising Polymerase Cham Reaction (PCR).

5. A composition of matter comprising DNA, substantially free of added nucleases, and containing less tihan about 3% by weight RNA and a compaction agent selected from the group consisting of: quaternary ammonium polyamines, the compounds of FTGS. 1-3 and PTCs.

6. The method of Claim 5 wherein the polyamine comprises a polyquaternary ammonium compound.

7. The method of Claim 19 wherein the enzyme is selected from the group consisting of reverse transcriptase, helicase, DNA polymerase, RNA polymerase, and nucleic acid modifying enzymes retaining substantial activity above 60 degrees C.

8. The method of Claim 1 wherein the RNA comprises an RNA selected from the group consisting of messenger RNA, microRNA, ribozymes, riboswitches, and ribosomal RNAs.

9. The method of Claim 2 wherein the compaction agent comprises a compound having a structure of Fig. I.

10. The method of Claim 2 wherein the compaction agent comprises a compound selected from the group consisting of compounds of Table B.

11. A method of Claim 1 comprising preparing substantially purified DNA without the use of nucleases or proteases, or organic solvent extraction, comprising adding an effective amount of a compaction agent selected from the group consisting of: quaternary ammonium polyamines, the compounds of FIGS. 1-3, and PTCs to a lysate containing DNA and RNA to selectively precipitate from said lysate, plasmid-DNA, chromosomal-DNA, or chromosomal DNA fragments having a content of less than 40% by weight RNA.

12. A method of Claim 1 for making an RNA product comprising less than 1 wt% gPNA content.

13. A method of claim 1 for preparing a substantially purified RNA having a genomic DNA content of less than 1 % and a substantially purified DNA having an RNA content of less than 10% without the use of nucleases or proteases or organic solvent extraction, comprising adding an effective amount of a compaction agent selected from quaternary ammonium polyamines, and Phase Transfer Catalysts (PTCs) to a lysate containing DNA and RNA to selectively precipitate from said lysate, pϊasimd-DNA, chromosomal-DNA, and/or chromosomal DNA fragments.

14. A mixture comprising RNA, DNA, a compaction precipitation agent selected from compounds of Figure 1 , and a reverse transcriptase or DNA polymerase enzyme.

15. The mixture of Claim 14 wherein the mixture comprises messenger-RNA (mRNA) and DNA monomer nucleotides.

16. The mixture of Claim 14 wherein the DNA comprises a cDNA copy of the mRNA, reverse- transcribed into DNA form by a template-guided polymerization.

17. The mixture of Claim 14 wherein the compaction agent is selected from the group consisting of poly quaternary ammonium compounds.

18. A method of purifying a protein from a mixture comprising protein and nucleic acid, said method comprising in combination the steps of:

A. adding a compaction agent of selected from the group consisting of the compounds of Figures 1-3 to effect compaction precipitation to form a nucleic-acid-depleted supernatant; and

B. separating the resulting purified protein from the resulting nucleic-acid-depleted supernatant,

19. A method ofassaying DNA or RNA in a mixture comprising DNA and RNA, said method comprising in combination the steps of:

A. adding a compaction agent to effect compaction precipitation; and

B. adding an enzyme to catalyze a reaction; and

C. detecting a product of the reaction catalyzed by the enzyme, to assay DNfA or RNA; wherein the compaction agent is selected from the compounds of Figures 1-3.

20. A method of preparing substantially purified RNA without the use of nucleases or proteases, or organic solvent extraction, comprising adding an effective amount of a compaction agent selected from the group consisting of: quaternary ammonium polyamines, the compounds of FIGS. 1-3, and PTCs to a lysate containing DNA and RNA to selectively precipitate from said tysate, plasmid-DNA, chromosomal-DNA, or chromosomal DNA fragments having a content of less than 40% by weight RNA.