Process for the separation of double-stranded/single-stranded nucleic acid structures

• US 6,180,778 B1
• Filed: 08/08/1996
• Issued: 01/30/2001
• Est. Priority Date: 02/11/1994
• Status: Expired due to Term

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Gauch, Simone, Bastian, Helge, Colpan, Metin, Feuser, Petra
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/687,610
• Time in Patent Office: 1,636 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.4, 536/25.41
• US Class Current: 536/25.4
• 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

Abstract

The present invention pertains to a process for the chromatographic separation of nucleic acid mixtures into their double-stranded and single-stranded nucleic acid fractions by simultaneously absorbing said nucleic acids as a whole to a mineral support, followed by separation into double-stranded and single-stranded nucleic acids by fractional elution, or by selectively absorbing double-stranded or single-stranded nucleic acid of a liquid sample to a mineral support, as well as solutions and a kit for performing the process according to the invention.

Claims

1. A process for the separation of single-stranded nucleic acids from double-stranded nucleic acids by treatment of a biological source, thereof, said treatment comprising the steps of:

a) applying to a first mineral support an aqueous solution containing a sample of said source under conditions whereby said first mineral support adsorbs only one of said single- or double-stranded nucleic acids followed by, optionally, washing said first mineral support; and

b) applying to a second mineral support the other of said single- or double-stranded nucleic acids, which was not adsorbed by the first mineral support, in an aqueous solution containing materials with alcohol groups.

2. The process of claim 1 wherein

i) the applying step to the first mineral support comprises adding to said aqueous solution salts and materials carrying alcohol groups in amounts such that the single-stranded, but not the double stranded, nucleic acids are adsorbed on the first mineral support, followed by, optionally, washing said first mineral support,

ii) the double-stranded nucleic acids, which were not adsorbed on the first mineral support, are applied to the second mineral support in the presence of materials with alcohol groups in amounts such that the double-stranded nucleic acids are adsorbed on the second mineral support, followed by, optionally, washing said second mineral support, and

iii) the single-stranded and double-stranded nucleic acids are eluted from the first and second mineral supports.

3. The process of claim 1, wherein

i) the applying step to the first mineral support comprises adding said aqueous solution with materials which complex alkaline-earth metal ions, in the absence of materials with alcohol groups, such that double-stranded, but not single-stranded nucleic acids are absorbed on the first mineral support,

ii) the single-stranded nucleic acids, which were not absorbed on the first mineral support, are applied to the second mineral support in the presence of materials with alcohol groups in amounts such that the single-stranded nucleic acids are absorbed on the second mineral support, followed by optionally, washing said second mineral support, and

iii) the double-stranded and single-stranded nucleic acids are eluted from the first and second mineral supports.

4. The process of claim 1 wherein

i) the applying step to the first mineral support comprises adding to said aqueous solution wetting, washing, or dispersing agents, in the absence of materials with alcohol groups, such that said double-stranded nucleic acids are absorbed on the first mineral support, followed by, washing said first mineral support,

ii) the single-stranded nucleic acids, which were not absorbed on the first mineral support, are applied to the second mineral support in the presence of materials with alcohol groups in amounts such that the single-stranded nucleic acids are absorbed on the second mineral support, followed by optionally, washing said second mineral support, and

iii) eluting the single-stranded and double-stranded nucleic acids from the first and second mineral supports.

5. The process of claim 1 wherein

i) the applying step to the first mineral support comprises adding to said aqueous solution salts and materials with alcohol groups in amounts such that both the single-stranded and double-stranded nucleic acids are adsorbed on the first mineral support,

ii) one of the single- or double-stranded nucleic acids is, selectively, first eluted from the first mineral support, followed by eluting the other of the single- or double-stranded nucleic acids, and

iii) the one of the single-.or double-stranded nucleic acids, which was first eluted from the first mineral support, is applied to the second mineral support under conditions whereby the nucleic acids first eluted from the first mineral support are adsorbed on the second mineral support, followed by eluting the nucleic acids from the second mineral support.

6. The process according to claim 1, further comprising the step, whereby, prior to applying the sample to the first mineral support, cells in said source containing the nucleic acids are lysed with chaotropic substances present in concentrations of from 0.1 to 10 M.

7. The process according to claim 2, wherein the salts in said aqueous solution, applied to the first mineral support, are selected from the group consisting of sodium perchlorate, guanidinium chloride, guanidinium isothiocyanate/guanidinium thiocyanate, sodium iodide, potassium iodide, and combinations thereof, in concentrations of from 0.1 to 10 M.

8. The process according to claim 3, wherein the salts in said aqueous solution, applied to the first mineral support, are selected from the group consisting of sodium perchlorate, guanidinium chloride, guanidinium isothiocyanate/guanidinium thiocyanate, sodium iodide, potassium iodide, and combinations thereof, in concentrations of from 0.1 to 10 M.

9. The process according to claim 4, wherein the salts in said aqueous solution, applied to the first mineral support, are selected from the group consisting of sodium perchlorate, guanidinium chloride, guanidinium isothiocyanate/guanidinium thiocyanate, sodium iodide, potassium iodide, and combinations thereof, in concentrations of from 0.1 to 10 M.

10. The process according to claim 5, wherein the salts in said aqueous solution, applied to the first mineral support, are selected from the group consisting of sodium perchlorate, guanidinium chloride, guanidinium isothiocyanatelguanidinium thiocyanate, sodium iodide, potassium iodide, and combinations thereof, in concentrations of from 0.1 to 10 M.

11. The process according to claim 2, wherein the salts in said aqueous solution, applied to the first mineral support, are selected from the group consisting of sodium chloride, lithium chloride, potassium chloride, sodium acetate, magnesium chloride, urea, and combinations thereof, in concentrations of from 0.1 to 10 M.

12. The process according to claim 3, wherein the salts in said aqueous solution, applied to the first mineral support, are selected from the group consisting of sodium chloride, lithium chloride, potassium chloride, sodium acetate, magnesium chloride, urea, and combinations thereof, in concentrations of from 0.1 to 10 M.

13. The process according to claim 4, wherein the salts in said aqueous solution, applied to the first mineral support, are selected from the group consisting of sodium chloride, lithium chloride, potassium chloride, sodium acetate, magnesium chloride, urea, and combinations thereof, in concentrations of from 0.1 to 10 M.

14. The process according to claim 5, wherein the salts in said aqueous solution, applied to the first mineral support, are selected from the group consisting of sodium chloride, lithium chloride, potassium chloride, sodium acetate, magnesium chloride, urea, and combinations thereof, in concentrations of from 0.1 to 10 M.

15. The process according to claim 2, wherein said materials containing alcohol groups are lower aliphatic alcohols in concentrations of from 1 to 90% by volume, and said salts, are present in concentrations of from 1 to 10 M.

16. The process according to claim 3, wherein said materials containing alcohol groups are lower aliphatic alcohols in concentrations of from 1 to 90% by volume, and said salts are present in concentrations of from 1 to 10 M.

17. The process according to claim 4, wherein said materials containing alcohol groups are lower aliphatic alcohols in concentrations of from 1 to 90% by volume, and said salts, are present in concentrations of from 1 to 10 M.

18. The process according to claim 5, wherein said materials containing alcohol groups are lower aliphatic alcohols in concentrations of from 1 to 90% by volume, and said salts, are present in concentrations of from 1 to 10 M.

19. The process according to claim 1, wherein said first and second mineral supports are porous or non-porous and comprised of metal oxides or mixed metal oxides, silica gel, glass particles, powdered glass, quartz, alumina, zeolites, titanium dioxide, or zirconium dioxide, the particle size of the mineral supports is from 0.1 μ

m to 1000 μ

m, and the pore size of porous mineral supports is from 2 to 1000 μ

m.

20. The process according to claim 1, wherein said biological source is selected from cell cultures, microorganisms, plants, or biological samples containing nucleic acids which are mixtures obtained from enzymatic reactions.

21. The process according to claim 1, wherein the single- or double-stranded nucleic acids obtained, thereby are respectively purified by chromatographic steps.

22. The process according to claim 3 wherein the complexes comprise alkaline earth metal ions bound to ethylenediaminetetraacetic acid (EDTA) or EGTA.

23. The process of claim 4 wherein said wetting, washing or dispersing agent is a sarcosinate.

24. The process according to claim 1, wherein said single-stranded nucleic acid is RNA and said double-stranded nucleic acid is DNA.

25. The process according to claim 2, wherein said single-stranded nucleic acid is RNA and said double-stranded nucleic acid is DNA.

26. The process according to claim 15, wherein lower aliphatic alcohols are selected from the group consisting of methanol, ethanol, isopropanol, butanol, and pentanol, and said salts are selected from the group consisting of NaCl, KCl, LiCl, MgCl₂, and NaAc.

27. The process according to claim 16, wherein lower aliphatic alcohols are selected from the group consisting of methanol, ethanol, isopropanol, butanol, and pentanol, and said salts are selected from the group consisting of NaCl, KCl, LiCl, MgCl₂, and NaAc.

28. The process according to claim 17, wherein lower aliphatic alcohols are selected from the group consisting of methanol, ethanol, isopropanol, butanol, and pentanol, and said salts are selected from the group consisting of NaCl, KCl, LiCl, MgCl₂, and NaAc.

29. The process according to claim 18, wherein lower aliphatic alcohols are selected from the group consisting of methanol, ethanol, isopropanol, butanol, and pentanol, and said salts are selected from the group consisting of NaCl, KCl, LiCl, MgCl₂, and NaAc.

30. The process according to claim 20, wherein said biological source is selected from blood, plasma, serum, urine, feces, bacteria, viruses, mixtures obtained by (i) enzymatic in vitro transcription, cDNA synthesis, or reverse transcription followed by (ii) PCR synthesis.