Device and process for automated extraction of nucleic acids

Abstract

A device and method are useful for automated extraction of nucleic acids. The device can include a body that can be immersed partly or completely in a reaction cavity, where the part immersed in the reaction cavity has a non-smooth surface. After lysis, an organic substance, preferably alcohols or ketones, can be mixed with a biological sample. This mixture can be brought into contact with a material with a non-smooth surface. Under these conditions, nucleic acids may be adsorbed on the surface of the material being used. Washing steps may be carried out. After drying, the adsorbed nucleic acid may be detached from the material by adding water or a buffer of low salt concentration, whereupon it can be used for downstream applications.

Claims

1. A device for automated extraction of a nucleic acid, the device comprising: at least one body that can be immersed partly or completely in a reaction cavity containing a nucleic acid, wherein at least a part of the at least one body immersed in the reaction cavity has a rough or structured surface, wherein the rough or structured surface is configured to bind to the nucleic acid during automated extraction of the nucleic acid and when the rough or structured surface is immersed in the reaction cavity, and wherein the at least one body comprises a smooth hollow body on which a rough or structured object is mounted externally, the rough or structured object being a ring or a sleeve mounted externally on the smooth hollow body.

2. The device according to claim 1, wherein the rough or structured object mounted externally on the smooth hollow body of the device is a rough or structured polymer material, a composite material with rough or structured surface or a material produced by 3D printing.

3. The device according to claim 1, wherein the hollow body is configured to receive a liquid disposed in the reaction cavity.

4. An instrument for automated extraction of nucleic acid, the instrument comprising: at least one device according to claim 1.

5. The instrument according to claim 4, wherein the instrument is an automated pipetting system or an automated extraction system.

6. A method for automated extraction of a nucleic acid, the method comprising: providing a nucleic acid in a reaction cavity; immersing the device according to claim 1 in the reaction cavity; and binding the nucleic acid on the rough or structured surface of the device.

Description

EXEMPLARY EMBODIMENT

(1) Automated Extraction of Nucleic Acid from NIH 3T3 Cells by Means of the Inventive Method and Using a Modified Pipette Tin as Well as Using a Commercially Available Automated Extraction System

(2) Variant A: Semiautomated Extraction Process (Sample Lysis Takes Place Separately)

(3) The InnuPure C16 (Analytik Jena AG) was used as an example of a standard automated extraction system. This system is a magnetic-particle-based extraction system, which was used outside its normal purpose to perform the inventive method. At the lower end of the pipette tips used for the InnuPure C16 automated system, a ring was slipped on externally in such a way that the pipetting function was not impaired. This externally slipped-on ring consists of a polymer and has a structured surface. The combination of hollow body and ring fastened thereon forms the means for performing the inventive method.

(4) Different quantities of NIH 3T3 cells were used for the extraction of nucleic acids. The extraction chemistry used for isolation of the nucleic acids was obtained in part from the commercial extraction kit known as innuPREP Blood DNA Kit/IPC 16X (Analytik Jena AG). Using a lysis buffer (Lysis Solution CBV) as well as Proteinase K, the cells were lysed at 60 C. for 15 minutes in a 2.0-mL reaction vessel. This lysis was not performed in the automated extraction system. Subsequently, the automated method of the Innupure C16 was used for purification of the nucleic acids. The solutions needed for extraction were present in a prefilled deep-well plate. The lysates described hereinabove were introduced into cavities filled with 400 L isopropanol. The pipette tips equipped with the ring (the inventive means) were subjected to 80 cycles of vertical immersion movement in these cavities, each including a waiting period of 2 s at the bottom of the cavity for incubation. Then the pipette tips modified with the ring were successively immersed 10 times each in three further cavities, which contained the alcoholic washing buffer (Washing Solution LS, 80% ethanol, 80% ethanol).

(5) Following the last washing step, the ring on the hollow body was dried for 10 minutes outside the cavity, and in this way the remaining ethanol was removed. The nucleic acids were eluted by 30 repetitions of immersion in and removal from 200 L Elution Buffer, which had been previously adjusted to a temperature of 50 C. by the instrument. In the same cavity, a mixing step then took place by means of 80 cycles of pipetting of 100 L at 40 C. The inventive double function of the inventive means was used for this purpose.

(6) The method is extremely easy to perform and thereby is extremely fast. Compared with the standard method of nucleic acid extraction with the InnuPure C16 and the use of magnetic particles for binding the nucleic acids, the time savings is greater than 50%.

(7) The isolated nucleic acid was detected by means of spectrophotometric measurement combined with gel-electrophoretic visualization in an agarose gel.

(8) Results of the spectrophotometric measurement:

(9) TABLE-US-00001 Con- Ratio Ratio centration Yield A.sub.260: A.sub.260: Sample (ng/L) (g) A.sub.280 A.sub.230 1 5 10.sup.5 NIH 3T3 cells 72.52 14.5 1.79 1.53 2 5 10.sup.5 NIH 3T3 cells 64.11 12.8 1.96 1.58 3 2.5 10.sup.5 NIH 3T3 cells 45.19 9.0 1.74 1.41 4 2.5 10.sup.5 NIH 3T3 cells 32.88 6.8 1.91 1.29 5 1.25 10.sup.5 NIH 3T3 cells 19.4 3.9 1.8 1.1 6 1.25 10.sup.5 NIH 3T3 cells 10.47 2.1 1.76 1.05 7 0.62 10.sup.5 NIH 3T3 cells 5.65 1.1 1.34 0.76 8 0.62 10.sup.5 NIH 3T3 cells 5.84 1.2 1.9 0.7

(10) As the results show, it is possible with the inventive means, solely by using standard extraction chemistry and commercially available extraction platforms, to bind and to isolate nucleic acids. It is evident that the yields are extremely high and that graduations can be observed in the yields depending on the cell quantities used.

(11) FIG. 2 shows the separation of the isolated nucleic acids by gel electrophoresis. It illustrates the nucleic acid isolated by means of the inventive method and separated electrophoretically in an 0.8% agarose gel. The samples were applied from left to right, beginning with sample 1. The applied volume was 5 L.

(12) Variant B: Fully Automated Extraction Process (Sample Lysis Takes Place in the Instrument)

(13) In a further embodiment, lysis of the sample likewise takes place in an automated process. Thus only the sample and the Proteinase K must be added by the user, while the further preparation takes place by a completely automated process using the technique of the InnuPure C16. For lysis of the sample, it is heated by the Innupure C16 to 50 C., and lysis is further intensified by 250 cycles of filling and emptying of the pipette. Thereafter 400 L isopropanol from a prefilled cavity is introduced into the cavity containing the lysate by the pipetting function of the hollow body. All further steps took place as described hereinabove.

(14) TABLE-US-00002 Concentration Yield Ratio Ratio Sample (ng/L) (g) A.sub.260:A.sub.280 A.sub.260:A.sub.230 1 2.5 10.sup.5 NIH 33.07 6.6 1.72 1.14 3T3 cells 2 2.5 10.sup.5 NIH 34.02 6.8 1.62 1.13 3T3 cells

(15) FIG. 3 shows the separation of the isolated nucleic acids by gel electrophoresis. It illustrates the nucleic acid isolated by means of the inventive method and internal lysis then separated electrophoretically in an 0.8% agarose gel. The samples were applied from left to right, beginning with sample 1. The applied volume was 5 L.

(16) FIG. 1 is an exemplary embodiment of the ring to be slipped onto a hollow body. The illustration is highly enlarged.

(17) It illustrates an exemplary embodiment of the ring just as it can be used for nucleic acid extraction according to the inventive method. This shaped body with non-smooth surface can be slipped onto any appropriate commercially available pipette tips, in such a way that it is then disposed in the last bottom third of the tip.