Nucleic acid processing kits and devices

• US 9,279,121 B2
• Filed: 11/10/2008
• Issued: 03/08/2016
• Est. Priority Date: 05/19/2003
• Status: Expired due to Fees

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Hartshorn, Cristina, Wangh, Lawrence J
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/292,037
• Publication Number:
  US 20090111170A1
• Time in Patent Office: 2,675 Days
• Field of Search: 435/6.1, 435/6.12
• US Class Current: 1/1
• 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: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• 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

Biological samples containing nucleic acids, RNA and DNA, are freed from bound proteins by incubation with a chaotropic agent such as a guanidium salt, and the mixture is readied for further processing by dilution of such agent to a concentration below 0.05 M without physical isolation of RNA and DNA from one another or from other components of the reaction mixture. Methods include such preparation and further processing, such as amplification and detection, which may be performed in a single container. Chaotropic agent may be supplied as a dried reagent adhered to a container. Kits may include such reagents, alone or with amplification reagents.

Claims

1. A method for enzymatically synthesizing cDNA from a biological sample, comprising:

(a) combining said biological sample with a disruption reagent in a container to produce a mixture comprising at least 2M chaotropic agent;

(b) diluting said mixture with at least one aqueous reagent to reduce the concentration of said chaotropic agent to less than 0.05 M; and

(c) incubating at least a portion of said mixture with reverse transcriptase.

2. The method according to claim 1, wherein the mixture is diluted to reduce the concentration of chaotropic agent to less than 0.01M.

3. The method according to claim 1, wherein said mixture includes at least one component selected from the group consisting of a cell-lysing detergent, a reducing agent, a water-miscible solvent, a chelating agent, and a neutralizing buffer.

4. The method according to claim 1, wherein dilution is accomplished by serial addition of at least two aqueous reagents.

5. The method according to claim 1, further comprising amplification of said cDNA.

6. The method according to claim 5, wherein said amplification is a polymerase chain reaction process.

7. The method according to claim 1, wherein an enzyme for performing said reverse transcriptase is included in said at least one aqueous reagent.

8. The method according to claim 1, wherein the step of diluting and/or the incubating is performed in said container.

9. The method according to claim 8 wherein steps (a), (b), and (c) are carried out in said container.

10. The method according to claim 1, to wherein the container is selected from the group consisting of a tube, a microtiter plate and a microfluidic device.

11. The method according to claim 1, further comprising a step between steps (a) and (b) of heating said mixture.

12. The method according to claim 11, wherein said heating is sufficient to at least partially dry the mixture.

13. The method according to claim 1, wherein said disruption reagent is a dry reagent.

14. The method according to claim 13, wherein said dry reagent is prepared from a mixture that includes a water-miscible solvent.

15. The method according to claim 13 wherein said dry disruption reagent is prepared from a mixture that includes at least one component selected from the group consisting of a cell-lysing detergent, a reducing agent, a chelating agent and a neutralizing buffer.

16. The method according to claim 13, wherein said dry reagent is adhered to a surface of a container.

17. The method according to claim 16, wherein said surface is the inner surface of a tube, a tube cap, a wall of a microtiter plate or a mictrotiter plate cover.

18. The method according to claim 1, wherein said sample comprises between a fraction of a cell and 200 cells.

19. The method according to claim 1, wherein the amount of disruption reagent is sufficient to provide a 2 M to 8 M concentration of the chaotropic agent in a volume of 20 to 50 nl.

20. The method according to claim 1, wherein said chaotropic agent denatures or degrades all proteins, including nucleases, to produce a disrupted sample containing RNA freed from bound proteins and inactivating nucleases.

21. The method according to claim 1, wherein combining said biological sample with a disruption reagent results in no more than twenty-five percent loss of RNA.