Isolation and amplification of nucleic acid materials

• US 20010021518A1
• Filed: 01/12/2001
• Published: 09/13/2001
• Est. Priority Date: 02/14/1996
• Status: Active Grant

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Inventors: Sol, Cornelis Johannes Andreas, Boom, Willem Rene, Beld, Marcellinus Gualbertus Hubertus Maria, Goudsmit, Jaap
• Granted Patent: US 7,238,530 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• 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 material from double stranded nucleic acid material comprising contacting a mixture of the both with a liquid comprising a chaotropic agent and a nucleic acid binding solid phase, whereby the liquid has a composition such that double stranded nucleic acid binds to the solid phase and a substantial amount of single stranded nucleic acid does not and separating the solid phase from the liquid.

2. A method according to

claim 1

whereby the liquid comprises at least 100 mM EDTA and comprising a guanidinium salt, preferably guanidinium thiocyanate as a chaotropic agent.

3. A method according to

claim 1

or

2

, whereby the solid phase is silicium based.

4. A method according to

claim 3

whereby the solid phase is silica.

5. A method according to

claim 4

whereby the silica is in the form of particles having a size between 0.05 and 500, preferably 0.1 and 200 μ

m.

11. A method for isolating and amplifying single stranded nucleic acid material originally present in a mixture of nucleic acids comprising subjecting the mixture to a method according to anyone of claims 1-7 followed by subjection of the isolated material to a method according to anyone of claims 8-10.

6. A method according to anyone of the aforegoing claims whereby the solid phase is separated from the supernatant by centrifugation.

7. A method for isolating single stranded nucleic acid material from a mixture of nucleic acid material, comprising the steps of subjecting the mixture to a method according to anyone of the aforegoing claims and treating the supernatant containing the single stranded nucleic acid material with a second liquid comprising a chaotropic agent and a second nucleic acid binding solid phase, whereby the second liquid has a compositon such that the resulting mixture of supernatant and second liquid allow for binding of the single stranded nucleic acid material to the second solid phase.

8. A method for amplifying single stranded nucleic acid material comprising the steps of hybridizing the single stranded nucleic acid with primers and elongating the probes using an enzyme which adds nucleotides to the primer sequence using the hybridized single strand material as a template, whereby at least one primer comprises a random hybridizing sequence and an amplification motif.

9. A method according to

claim 8

whereby at least one primer comprising a random hybridization sequence and an amplification sequence further comprises a label.

10. A method according to

claim 8

or

9

whereby at least one primer comprising a random hybridizing sequence and an amplification motif further comprises a direct sequencing motif.

12. A method according to any one of the aforegoing claims whereby the single stranded nuclec acid material comprises mRNA.

13. A method according to

claim 12

whereby the mRNA is converted into cDNA.

14. A method according to anyone of the aforegoing claims comprising a gel electrophoresis step.

15. A method according to anyone of the aforegoing claims followed by a sequencing step.