Method of isolating plasmid DNA

Details Method of isolating plasmid DNA Method of isolating plasmid DNA

2001-06-08

2003-04-15

Fredman, Jeffrey (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S022100, C536S025400

Reexamination Certificate

active

06548253

ABSTRACT:

The invention relates to a method of isolating plasmid DNA from microorganism cultures with the aid of solid-phase bodies.
In molecular biology, microorganisms (for example Escherichia coli, Salmonella typhimurium, etc.) have long been used for cloning experiments. To this end, extrachromasomal DNA, so-called plasmid DNA, is, employed as a vehicle for the specific DNA fragments to be cloned. It is an everyday task for the person skilled in the art to find the correct bacterial population with the desired plasmid clone. Various chemical methods for the isolation of plasmid DNA from microorganisms have been described. It is a common feature of all these methods that the microorganisms are firstly separated off by centrifugation in order to remove the nutrient medium. The nutrient medium must be removed as quantitatively as possible, and the microorganism precipitate obtained must be resuspended completely in a resuspension buffer.
The present invention has the object of providing a method of isolating plasmid DNA from microorganisms which does not require a centrifugation step for the removal of the nutrient medium.
The invention relates to a method of isolating plasmid DNA from microorganism cultures with the aid of solid-phase bodies which is characterised in that
a) the microorganism culture is brought to an acidic pH, mixed with the solid-phase bodies, incubated and separated off,
b) the microorganisms immobilized on the solid-phase bodies are resuspended, lysed, mixed with a neutralising binding buffer and incubated, and the solid-phase bodies are separated off and discarded,
c) the supernatant is again mixed with solid-phase bodies, incubated and subsequently separated off, and the plasmid DNA is eluted from the solid-phase bodies using an elution buffer.
Suitable solid-phase bodies are silica gels, silicates or glass-like materials, preferably magnetic solid-phase bodies having a silica-gel surface, in particular magnetic silica particles.
Surprisingly, it has been found that, under certain buffer conditions, bacteria bind non-specifically to magnetisable solid-phase bodies having a silica-gel surface and can be separated off magnetically directly from the nutrient medium. It has furthermore been found that the bacteria collected in this way can be resuspended, lysed and likewise separated off as a compact magnetic precipitate together with the genomic DNA, leaving the plasmid DNA in the supernatant. The invention is distinguished by the fact that it makes all method steps of the plasma isolation automatable.
The method according to the invention is carried out, for example, by adjusting a bacterial culture which has been incubated overnight in a growth medium to an acidic pH with the aid of buffers or acids. Magnetic particles are subsequently added, the mixture is incubated for a few minutes, and the particles are separated off in a magnetic field. The supernatant is discarded, and the bacteria adhering to the particles are resuspended in a resuspension buffer. A lysis buffer is then added, mixed and incubated for a few minutes at room temperature; a neutralising binding buffer (N-binding buffer) is added and mixed, and the particles are separated off in a magnetic field. The supernatant is transferred into a fresh reagent vessel, particles are added, mixed and separated off in a magnetic field. The particles with the bound plasmid DNA are washed with washing buffer, and finally the plasmid DNA is eluted with elution buffer.
The microorganism cultures are preferably
E. coli
or
E. coli
mutants, such as W3110, JM109, RR1, XL-1 Blue, inter alia. These cultures are generally incubated overnight at 37° C., for example in an LB medium
(10
g of Trypton, 5 g of yeast extract, 10 g of common salt per litre).
A culture prepared in a corresponding manner is adjusted to a pH in the range from 1 to 4, preferably pH 2, with the aid of buffers or acids. Suitable buffers are those which have an adequate buffer capacity in this pH range, for example formate, acetate or citrate buffer, or also acids, such as hydrochloric acid. 1 ml of the bacterial culture is acidified, for example using 0.1 ml of 1N hydrochloric acid, and mixed with 5-50 &mgr;l of a suspension (50 mg/ml) of magnetic silica particles, incubated for 1 minute and separated off in a magnetic field. It has been found that 5-15 &mgr;l of the particle suspension are generally sufficient to separate off more than 50% of the bacteria. The number of particles used here exceeds the number of bacteria approximately by a factor of 10-30.
The bacteria adhering to the particles are resuspended in a resuspension buffer. The buffer should be capable of maintaining a pH in the range from 7 to 9; a suitable resuspension buffer consists, for example, of tris-HCl, EDTA and RNase A.
A lysis buffer is added to the resuspended bacteria, mixed and incubated at room temperature for a few minutes. The lysis buffer consists, for example, of sodium hydroxide solution and SDS. A neutralising binding buffer (N-binding buffer) is subsequently added and mixed, and the particles are separated off in a magnetic field and discarded. The N-binding buffer should maintain a pH in the range from 4 to 6; a suitable N-binding buffer consists, for example, of a guanidinium salt and potassium acetate. However, the two buffer components can also be employed separately and successively (see Example 2).
The supernatant is transferred into a fresh reagent vessel, further particles are added and mixed, and the particles loaded with the plasmid DNA are separated off in a magnetic field and washed with washing buffer. This buffer should buffer in the pH range from 5 to 7, with the DNA remaining bound to the particles. A suitable washing buffer consists, for example, of tris-HCl and EDTA.
Subsequent elution of the plasmid DNA is carried out using an elution buffer. The elution buffer used for this purpose should maintain a pH in the range from 7.5 to 9.5, preferably from 8 to 9. Examples of suitable buffers are tris-HCl buffer, tricine, bicine and other buffers which buffer in this pH range, preferably tris-HCl. If desired, the elution buffer may contain chelating agents, such as EDTA, and/or other substances. The buffer concentration should be from 5 to 10 mM, preferably about 10 mM. If desired, the buffer may also contain small amounts of EDTA, for example 1 mM. The nucleic acid eluted in this way can be employed directly, without further purification steps, for molecular-biological applications, such as, for example, for amplification reactions (PCR, NASBA).


REFERENCES:
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patent: 5075430 (1991-12-01), Little
patent: 5378621 (1995-01-01), Lawlis, Jr. et al.
patent: 6255477 (2001-07-01), Kleiber et al.
patent: 4307262 (1994-09-01), None
patent: 19520398 (1996-12-01), None
patent: 9207863 (1992-05-01), None
Carter MJ et al. An inexpensive and simple method for DNA purifications on silica particles. Nucleic Acids. Res., 21(4): 1044, 1993.

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