JP2001526888A - Method for separating short and long chain nucleic acids - Google Patents

Abstract

(57) Abstract: The method according to the invention relates to the separation and purification of short and long chain nucleic acids from various starting materials, dissolving the nucleic acid containing material, incubating with an inorganic carrier material, the particle size of the carrier material. It is characterized in that it is applied to a membrane with a larger pore size and the nucleic acids are separated by elution from the carrier substance, whereby the carrier substance surprisingly remains on the membrane.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a simple and very fast method for separating and purifying large amounts of short and long nucleic acids from various biological and other starting materials. The method is important for many laboratories operating in the fields of biology, molecular biology, forensics, medicine, analysis and biochemistry. Therefore, the fields of application of the present invention are forensic medicine, medical diagnosis, molecular biology, biochemistry,
Genetic engineering and all other related fields.

All commercially available kits are based on the well-known principle of binding nucleic acids to inorganic carriers in the presence of various chaotropic salt solutions, and use finely ground glass powder (eg Glassmilk) as carrier material. BIO 101, La Jolla, CA), diatomaceous earth (Sigma) or silica gel (Diagen, DE 4139664 A1).

A practical method for separating nucleic acids for a number of different applications is shown in US Pat. No. 5,234,809 (Boom). It describes a method for separating nucleic acids from starting materials containing nucleic acids by incubating the starting materials with a chaotropic buffer and a solid phase bound to DNA. The chaotropic buffer allows dissolution of the starting material and binding of the nucleic acid to the solid phase. This method is suitable for separating nucleic acids from a small amount of sample, and has practical application particularly in the field of viral nucleic acid separation.

The object of the present invention is, inter alia, to provide a method which is very simple, fast and inexpensive to separate nucleic acids, in particular plasmid DNA, from a variety of starting materials and subsequently to obtain separated nucleic acids corresponding to high quality parameters of application. It is to develop.

This object is achieved according to the claims, ie according to the invention, by means of a combined batch column method, in which the binding to the inorganic carrier substance is carried out in a batch process and the bound nucleic acids are subsequently chromatographed. Is done.

The method according to the invention for separating and purifying short and long nucleic acids from various biological and other starting materials comprises: dissolving the material containing the nucleic acids, incubation with inorganic carrier substances, -Application to a membrane having a pore size larger than the particle size of the carrier substance,-separation of the nucleic acid from the carrier substance by elution, the carrier particles remaining on the membrane.

[0007] Suitable starting materials containing nucleic acids include complex biological systems, such as blood, tissue, urine or stool samples, optionally bound to solid substances. However, the method according to the invention is also suitable for the separation and purification of other starting materials such as bacterial lysates, short and long nucleic acids from plant components, or PCR adducts or DNA fragments from agarose gels.

[0008] Suitable carrier materials are silicon compounds such as silicon dioxide, silicic acid or silica gel. The silicon compounds used according to the invention have an average particle size of 7 nm to 5000 nm, preferably 7-40 nm or 500-500 nm.

[0009] Incubation with the carrier material following the dissolution of the starting material is performed in a buffer system with chaotropic salts. The chaotropic salt is preferably at a concentration of 1 to 10 M
For example, guanidine isothiocyanate, guanidine hydrochloride, lithium chloride, sodium perchlorate, sodium iodide and / or urea.

[0010] Detergents such as Triton X-100, Tween, N-lauryl sarkosyl, SDS and / or CTAB, and optionally proteolytic enzymes such as proteinases are used as components which promote dissolution.

According to the invention, for the separation of the inorganic carrier substance used, it is preferred to use a membrane having a pore size larger than the particle size of the carrier substance used, preferably in a centrifuge cartridge. In that case, the pore size of the membrane is determined according to the average particle size of the carrier material used. It is preferred to use a nylon or polysulfone membrane according to the invention.

The carrier on which the nucleic acid is immobilized is washed before or after application to the membrane. Subsequently, elution of the nucleic acid from the carrier is performed with a low ionic strength buffer, as will be clear to the skilled person.

[0013] It is generally self-evident that centrifugal membranes that can be effectively used for the purification of nucleic acids must have a pore size smaller than the particle size of the carrier material used. This is because inorganic particles having a smaller particle size than the pores of the centrifuge membrane are always centrifuged by the pores during the centrifugation step.

[0014] The present invention is based on the surprising confirmation that a combined batch column nucleic acid separation method incorporating inorganic carrier particles much smaller than the pore size of commercially available centrifugation columns is possible.

[0015] Surprisingly, such a composite method can also be realized with an inorganic carrier substance of 50 nm or less, so that the advantage of an extremely large binding surface of such particles can be exploited.

The process according to the invention, which is a combination of a batch process and a column process, is unlimited with respect to the volume of the starting material, since it utilizes any carrier material, and thus also small inorganic carrier particles and their very large specific surface area.

This means that the step of binding the plasmid DNA to the carrier material used does not have to be carried out in the column, so that conventionally it has to be increased according to the amount of starting material (eg bacterial lysate). Means that there is no longer any restriction on the stock solution.

The method results in very high yields of highly pure DNA and is extremely simple to carry out, for example in the case of plasmid DNA separation, which is much shorter than the widely used anion exchange system. Can be realized in time.

Next, the present invention will be described based on examples.

1. Microseparation of plasmid DNA Transfer 2 ml of bacterial overnight culture to a 2 ml Eppendorf reaction vessel and centrifuge at 12,000 rpm for 1 minute. Subsequently, the cell pellet is resuspended with 100 μl of solution I (Tris HCL, EDTA; glucose). Add 300 μl of Solution II (NaOH; SDS) and lyse the cells by briefly and carefully shaking the reaction vessel. The required neutralization reaction is performed by adding 300 μl of solution III (sodium acetate; guanidinium hydrochloride). Next, the chromosomal DNA and proteins are precipitated by a centrifugation step at 14,000 rpm for 5 minutes, and the supernatant obtained by centrifugation is then centrifuged to 20 μl of an inorganic carrier composed of silica nanoparticles (average particle size 40 nm).
Incubate with 1 suspension. Subsequently, the solution is transferred to a small centrifugation column (for example, Micro Spin Nylon or Micro Spin PSE; LIDA). The carrier particles to which the plasmid DNA is bound are transferred to the surface of the filter membrane by centrifugation at 12,000 rpm for 1 minute, whereupon they are washed by adding a washing solution (60% ethanol; NaCl) and another centrifugation step.
Repeat the washing step once more. Finally, residual ethanol is removed from the carrier material by centrifugation at 12,000 rpm for 2 minutes. 100 μl low salt buffer (eg 10 mM Tris
HCl) at 70 ° C and then centrifuged at 12,000 rpm for 1 minute to detach the bound plasmid DNA. The centrifuge contains the separated plasmid DNA.

FIG. 1 shows a gel electrophoresis diagram of isolated plasmid DNA (trace) of individual cell colonies.
Shown in

2. Medium volume isolation of plasmid DNA Transfer 25 ml of bacterial overnight culture to a 50 ml Flacon reaction vessel and centrifuge at 5,000 rpm for 10 minutes. Subsequently, the cell pellet is resuspended with 1 ml of solution I (Tris HCL, EDTA; glucose). Add 1.8 ml of Solution II (NaOH; SDS), lyse the cells by carefully shaking the reaction vessel briefly and incubating for 5 minutes. The required neutralization reaction is carried out by adding 1.8 ml of solution III (sodium acetate; guanidinium hydrochloride) and then incubating at ice temperature for 10 minutes. The chromosomal DNA and proteins are then precipitated by a centrifugation step at 5,000 rpm for 10 minutes, and the supernatant, which has been centrifuged transparently, is then incubated with a 150 μl suspension of an inorganic carrier consisting of silica nanoparticles (average particle size 40 nm). Subsequently, the solution is centrifuged (for example, Micro Spin Nylon; LIDA).
Company). The carrier particles to which the plasmid DNA is bound are transferred to the surface of the filter membrane by centrifugation at 5,000 rpm for 5 minutes, whereupon they are washed by adding a washing solution (60% ethanol; NaCl) and centrifuging again. Repeat the washing step once more. Finally at 5,000 rpm
The residual ethanol is removed from the carrier material by centrifugation for 10 minutes or incubation of the centrifugation column for 10 minutes at 37 ° C. 200 μl-300 μl of low salt buffer (eg,
Bound plasmid DNA is detached by adding 10 mM Tris-HCl) at 70 ° C and then centrifuging at 5,000 rpm for 5 minutes. The centrifuge is the separated plasmid DNA
including.

FIG. 2 shows a gel electrophoresis diagram of the isolated plasmid DNA (medium amount) of each individual cell colony.

[0024] 3. Comparison of required operating steps and time consumption between the method of the present invention and a commercially used anion exchanger-based plasmid DNA (medium) separation system

[Table 1]

[Brief description of the drawings]

FIG. 1 is a gel electrophoresis diagram of a separated plasmid DNA (trace amount).

FIG. 2 is a gel electrophoresis diagram of the separated plasmid DNA (medium amount).

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Claims (11)

[Claims] 1. dissolution of a material containing nucleic acids;-incubation with an inorganic carrier substance;-application to a membrane having a pore size larger than the particle size of the carrier substance;-carrier particles remaining on the membrane, elution of the carrier substance. For the separation and purification of short and long nucleic acids from various biological and other starting materials. 2. The starting material is a complex biological system, such as blood, tissue, urine or feces, bacterial lysate, plant components or PCR adducts or DNA fragments from agar gel, optionally combined with solid material. The method of claim 1, wherein: 3. The process as claimed in claim 1, wherein a silicon compound such as silicon dioxide, silicic acid or silica gel is used as the carrier substance. 4. The process according to claim 1, wherein the silicon compound used has an average particle size of 7 nn to 5000 nm, preferably 7 to 40 nm or 500 to 5000 nm. 5. The method according to claim 1, wherein the dissolution of the nucleic acid and the binding to the inorganic carrier substance are carried out by a combination of a chaotropic salt solution and a dissolved component. 6. The method according to claim 5, wherein guanidine isothiocyanate, guanidine hydrochloride, lithium chloride, sodium perchlorate, sodium iodide and / or urea are used as the chaotropic salt. 7. The method according to claim 5, wherein a detergent and / or a protease is used as the dissolving component. 8. The method according to claim 1, wherein a nylon or polysulfone membrane is used as the membrane. 9. The method according to claim 1, wherein the pore size of the membrane is determined according to the particle size of the carrier substance used. 10. The method according to claim 1, wherein the nucleic acid on which the carrier is immobilized is washed before or after the nucleic acid is applied to the membrane. 11. The method according to claim 1, wherein the elution of the nucleic acid from the carrier is performed using a buffer having a low ionic strength.