Isolation and amplification of nucleic acid materials

• US 7,238,530 B2
• Filed: 01/12/2001
• Issued: 07/03/2007
• Est. Priority Date: 02/14/1996
• Status: Expired due to Fees

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux SA (Compagnie Merieux Alliance SAS)
• Inventors: Goudsmit, Jaap, Sol, Cornelis Johannes Andreas, Boom, Willem Rene, Beld, Marcellinus Gualbertus Hubertus Maria
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Tung; Joyce
• Application Number:
  US09/760,085
• Publication Number:
  US 20010021518A1
• Time in Patent Office: 2,363 Days
• Field of Search: 435/6, 435/91.2, 536/25.4, 536/25.41, 536/26.6
• US Class Current: 436/6
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• 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 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium

Abstract

The invention relates to methods for easily separating single stranded nucleic acid material from double stranded nucleic acid material in a sample containing both. By the right choice of at lease one chaotropic agent, preferably a guanidine salt, at a selected concentration and other suitable conditions such a chelating agents, pH and the like, it is possible to bind double stranded material to a solid phase such as silica particles, whereas single stranded material will not bind under those circumstances. By separating the silica particles from the sample the double stranded nucleic acid material is removed. It can easily be eluted from the silica particles. In a second step the single stranded material may be bound to a solid phase by selecting a different set of conditions. The particles can again be separated from the sample and the single stranded material may now be eluted. For very efficent separations, the process may be repeated. Following the separation of the two kinds of nucleic acid, either kind may be amplified. Methods of amplification are provided which do not need sequence data of the material to be amplified. In these methods a primer will be provided with an amplification motif and a random hybridization motif.

Claims

1. A method for separating single stranded nucleic acid from double stranded nucleic acid, comprising the steps of:

contacting a mixture comprising both single stranded nucleic acid and double stranded nucleic acid with a first liquid comprising a chaotropic agent and a nucleic acid binding solid phase, wherein the first liquid has a composition such that the double stranded nucleic acid binds to the solid phase;

separating the solid phase from a supernatant containing the single stranded nucleic acid; and

contacting the supernatant with a second nucleic acid binding solid phase, and a second liquid consisting essentially of material selected from the group consisting of;

a) a chaotropic agent

b) a chaotropic agent and a chelating agent;

c) a chaotropic agent and divalent positive ions; and

d) a chaotropic agent, a chelating agent and divalent positive ions,

wherein the second liquid has a composition such that the resulting mixture of supernatant and second liquid allows for binding of the single stranded nucleic acid to the second solid phase.

2. The method according to claim 1, wherein the first liquid comprises a chaotropic agent in concentration between about 1–

10M, and a chelating agent, and has a pH between about 2 and 10.

3. The method according to claim 2, wherein the chelating agent is EDTA, which is present in a concentration between about 10 mM and 1M.

4. The method according to claim 3, wherein the first liquid comprises at least about 100 mM EDTA and guanidinium salt as a chaotropic agent.

5. The method according to claim 1, wherein the chaotropic agent is guanidinium thiocyante.

6. The method according to claim 5, whereby the first liquid has the constitution of a buffer prepared by dissolving about 120 g guanidinium thiocyanate in about 1 ml 0.2M EDTA (pH=8).

7. The method according to claim 1, wherein the solid phase is silicum based.

8. The method according to claim 7, wherein the solid phase is silica.

9. The method according to claim 8, wherein the silica is in the form of particles having a size between about 0.05 and about 500 micrometers.

10. The method according to claim 1, wherein the solid phase is separated from the supernatant by centrifugation.

11. The method according to claim 1, wherein the concentration of the divalent positive ions is the same as the concentration of the chelating agent.

12. The method according to claim 1, wherein the chelating agent is EDTA and the ions are Mg²⁺ ions.

13. The method according to claim 1, wherein the chaotropic agent is a guanidinium salt.

14. The method according to claim 1, wherein the second liquid has the constitution of a buffer prepared by dissolving about 120 g guanidinium isothiocyanate in about 100 ml 0.35M TRIS HCl (pH 6.4) and adding about 22 ml 0.2 M EDTA (pH 8.0) and about 9.1 g Triton X-100™

(polyethoxylated p-isooctyl-phenol), homogenizing the solution and adding MgCl₂ to a final concentration of about 0.25M.