(51) Int.Cl.7

DESCRIPTION

The present inventive relates to a reagent kit for isolating nucleic acids from a biological sample, comprising nucleic acid-containing compartments; a method for isolating nucleic acids from a biological sample, comprising nucleic acid-containing compartments; as well as a method for amplifying and/or determining the nucleic acids by means of polymerase chain reaction.

The polymerase chain reaction (PCR), described in the US-PS 4,683,195, is a simple method for replicating nucleic acids. This replication has made it possible to detect the tiniest amounts of nucleic acids. The method has a sensitivity of detection that is unparalleled to date in scientific analysis chemistry. Whereas in the past detection was not possible up to approximately 10⁶ molecules, it is now possible to detect amounts as small as about 10 to 100 molecules. A current and spectacular application of this sensitivity of detection is, for example, the identification of offenders in clearing up a crime. The PCR technology is an essential element in the creation of a "gene database", which has been discussed by the general public. PCR is also mandatory for detecting genetically modified food or for testing stored blood for viruses, like HIV or HCV.

In a very short period of time the PCR technology has established itself in the daily scientific work and, hence, represents a growth market. The prerequisite for the PCR method is a so-called "sample preparation", which releases the nucleic acids from a variety of widely varying material, like saliva, blood, fluids; organisms, such as bacteria, yeast, fungi; and also plant material, meat and other nucleic acid-containing samples, and which prepares in such a way that the PCR process can be carried out without any trouble. Currently this sample preparation is the bottleneck for the broad application, primarily due to the lack of automatable processes.

It is very important for automating the control process that the particles, which are used in the solid phase, are introduced either together with filtration or centrifugation in the sample preparation, or can be removed in the form of so-called magnetic particles by means of magnetic fields. The US 4,554,088 can be cited as a source for the comprehensive literature.

The literature describes a plethora of methods for the sample preparation with respect to the PCR. Some examples are the use of inorganic silicate surfaces as the adsorber for nucleic acids, which bind, according to EP 0 389 063, to the silica in the presence of chaotropic salts and can be isolated with this principle.

The US-PS 5,705,628 describes, as the adsorber for nucleic acids, magnetic particles having a carboxyl group-coated surface. The use of such particles is described in a multi-step process, where in a time staggered sequence a sample is treated with a lysis buffer in order to lyse the sample, then with the magnetic particle suspension and thereafter with a special buffer, which adjusts the polarity and the hydrophobicity of the resulting solution in such a way that the nucleic acids bind reversibly to the magnetic particles. In this case the buffer is a highly viscose buffer, which comprises polyethylene glycol (20%) together with 2.5 M of common salt (hereinafter called the PEG buffer).

After washing once or multiple times the magnetic particles and after resuspension of the magnetic particles in an elution buffer, the nucleic acid is desorbed and, following removal of the magnetic particles, can be removed. However, the process presents three problems with respect to an automatic control:

a) The pipetting steps, which are necessary in a time staggered sequence, are extremely time consuming for an automatic control process - for example, with a pipetting robot -, so that this method is not suited for a higher sample throughput.

b) The use of a highly viscous buffer requires for the automatic control process a considerable material cost in order to achieve the correspondingly necessary thorough mixing. Since the automatic control process uses chiefly 96‑well microtitration plates as the reaction vessel, an appropriate shaking or agitating device or other mechanical mixing must ensure uniform mixing in all of the vessels.

c) Another problem arises from the fact that the samples exhibit a wide range of characteristics. The known methods for sample preparation left much to be desired with respect to their effectiveness for many samples.

Therefore, the object of the present invention was to provide a possibility for designing the sample preparation so as to be more effective while at the same time avoiding the drawbacks associated with the state of the art. In particular, it was the object of the present invention to provide possibilities for carrying out the sample preparation and optionally also the subsequent PCR reaction by means of the use of pipetting robots and, to this end, to make it possible to simplify and automate the past methods.

This object was achieved, according to the invention, by means of a reagent kit, which is intended for isolating nucleic acids from a biological sample, comprising nucleic acid-containing compartments, and which is characterized in that the reagent kit comprises

a) negatively charged particles, composed of a polymer material,

b) a reagent I, comprising a mixture of a charged or uncharged detergent and an aliphatic alcohol, and/or a reagent II, comprising an aqueous solution of at least one chaotropic salt, and

c) a solution of protease.

The concept "biological, nucleic acid-containing compartments" is defined within the scope of the present invention as all structures in the samples, which are to be tested and from which nucleic acids have to be released by lysis. In particular, cells, including bacteria or yeast cells, as well as viruses are included in this definition.

The inventive reagent kit comprises, as the reagents I and/or II, compositions exhibiting surprisingly simple formulations, which can be used for an effective lysis of the samples - that is, the biological, nucleic acid-containing compartments. Moreover, the reagents I and II, contained in the inventive reagent kit, make it possible to bind immediately, following lysis, the released nucleic acids to the polymer particles without having to change the buffer and/or to add additional substances. By adding neutral cationic or anionic detergents in high concentrations together with aliphatic alcohols it is possible to achieve an especially efficient and comprehensive lysis, a procedure that holds true, in particular, for samples that contain yeast cells. If the reagent II, containing an aqueous solution of at least one chaotropic salt is used, it is possible to bring about an especially efficient lysis of samples, containing whole blood. For all other applications, the reagents I and II are just as suitable. Hence, the person skilled in the art can easily determine beforehand whether the one or the other reagent is better suited for the respective application.

The inventive reagent kit makes it possible to carry out the lysis of cells or viruses and to bind the nucleic acids to particles as well as to remove the particles from the sample solution in a quasi one step process by adding the sample substance to a mixture comprising the particles, the reagent I or II and the protease solution. Then after a suitable incubation the sample solution can be removed again while simultaneously leaving behind the nucleic acids, separated from the cells, in a form bound to the polymer particles.

In a preferred embodiment of the invention the reagent kit also contains additionally a wash buffer and/or elution buffer, in order to remove the nucleic acids from the polymer particles.

Therefore, in an especially preferred embodiment the inventive reagent kit contains all of the reagents for carrying the isolation of the nucleic acid from the samples. Thus, there is the possibility of holding, first of all, the reagents individually available - thus, in separate bottles - in the reagent kit and then producing a stable working solution just immediately before the application. This procedure has the advantage that a very wide variety of different lysis principles can be applied as a function of the sample. Thus, it was found surprisingly that a buffer with sodium dodecyl sulfate together with ethanol is especially advantageous for isolating nucleic acid from yeast, whereas a buffer with guanidinium thiocyanate is better suited for the lysis and the isolation of nucleic acids from whole blood.

Therefore, in a preferred design, the inventive reagent kit contains in separate bottles the reagents A) particles, B) reagent I with a mixture of detergent and alcohol, C) reagent II with high molecular salts of chaotropic compounds as well as D) a solution of protease.

In addition, the inventive reagent kit can also provide ready-for-use one or more working solutions, comprising mixtures of A, B and D as well as of A, C and D.

An inventive reagent kit, which contains both reagent I as well as reagent II, is suitable for the isolation of nucleic acids from a large plurality of samples. In this case the final production of the working solution is performed by the user as a function of the respective application. Of course, the scope of the present invention also includes a reagent kit that contains only one of the two reagents and is, therefore, especially suited for isolating, for example, nucleic acids from yeast-containing samples and/or whole blood. The application of one of the reagents in other samples shall not be restricted by these preferred applications.

As stated above, other components of the inventive reagent kit can be wash solutions for the particles. These wash solutions are poured into a separate bottle. In order to elute the nucleic acid from the particles, an elution buffer is required that should be distinguished, in particular, by its low ionic strength. This component can also be a part of the inventive reagent kit. Both the suitable wash solutions and the elution buffer are known, in principle, to the person skilled in the art.

The operating principle of the individual components of the inventive reagent kit is described below.

A sample is treated with a working solution, comprising the constituents A, B, D or A, C, D and incubated. In the individual case the incubation can take place at a higher temperature. The working solution simultaneously brings about the lysis of the sample, the release of the nucleic acids as well as the binding to the particles, which are a part of the working solution. The particles are separated; the clear supernatant is extracted and discarded. Then the particles are washed in a suitable wash solution. In this case owing to the ease of miscibility the alcoholic solutions in turn prove to be especially advantageous. In particular, mixtures of water and ethanol or in general mixtures of water and aliphatic alcohol in a ratio of 70 to 30 parts of water with 30 to 70 parts of alcohol yield, according to the invention, satisfactory results. The washing process is repeated once or several times as a function of the sample; and the washed and deposited particles are absorbed with a buffer of low ionic strength, so that the nucleic acids desorb and dissolve. Thereafter, additional processing - for example, for a PCR amplification - can take place.

In one preferred embodiment of the invention, the reagent kit contains, as the negatively charged particles, those particles that are composed of polystyrene or polyvinyl alcohol, where the particles of polyvinyl alcohol represent an especially preferred embodiment.

Even though in principle it is, of course, possible to use non-magnetically influencable particles, it is preferred to provide magnetically influencable particles, which during the isolation of the nucleic acids are especially easy to concentrate by way of a magnet in one location in the reactor vessel and from which the remaining sample solution is especially easy to separate.

Preferably the particles in the reagent kit exhibit an average diameter ranging from 0.1 to 100 mm and, in particular, from 1 to 10 mm. Particles of this diameter allow the nucleic acids to bind well and effectively.

In a preferred embodiment of the invention, the negative charge of the particles is based on the presence of the carboxyl groups on the surface of the particles. Particles, whose use is especially preferred within the scope of the present invention, are described, for example, in the EP 0 843 591.

Preferably the reagent I of the inventive reagent kit contains, as the detergent, sodium dodecyl sulfate or cetyl ammonium bromide (CTAB), preferably in an amount ranging from 1 to 10%, based on the reagent volume.

Reagent I contains, as the aliphatic alcohol, preferably ethanol, where a concentration of at least 40% by volume leads to good results and is, therefore, preferred.

Reagent II contains preferably aqueous solutions of guanidinium hydrochloride or guanidinium thiocyanate, where in turn concentrations of these salts in a range between 2 and 8 M are preferred.

The reagents I and II contain, if desired, additional customary and suitable buffers and/or auxiliary substances. Examples of buffer substances are Tris/HCl. Auxiliary substances may be, for example, complexing agents, like EDTA. The pH value of the reagents is adjusted preferably to approximately the physiological pH.

The protease, contained in the inventive reagent kit, serves to avoid trouble owing to the presence of proteins following lysis of the biological, nucleic acid-containing compartments. Preferably to this end the inventive reagent kit includes the protease K. In this case the amount of protease used during the nucleic acid isolation depends on the amount of existing cells and, thus, on the amount of proteins. Appropriate concentrations of protease can be easily determined by the person skilled in the art.

The inventive reagent kit makes it possible to isolate easily and quickly the nucleic acids from samples - in particular, for the next phase, in which a PCR reaction takes place. Since only the sample has to be added to, for example, a prepared working solution containing all of the necessary components, it is easy to remove, following separation of the particles by means, for example, of a magnet, the remaining sample solution; and the sample can be eluted, following an optional necessary washing, while at the same time isolating the nucleic acids. In this way a corresponding method can be easily automated. To this end, only about 4 pipetting steps are necessary up to the final recovery of the sample.

Therefore, an additional subject matter of the present invention is a method, which is intended for isolating nucleic acids from a biological sample, comprising nucleic acid-containing compartments, and which is characterized in that the sample is treated with negatively charged particles, composed of a polymer material, and with a reagent, comprising a mixture of a charged or uncharged detergent and an aliphatic alcohol, or comprising an aqueous solution of at least one chaotropic salt; then the particles are separated in a suitable way from the supernatant solution, are optionally washed; and then the nucleic acids, bound to the particles, are released from the particles by means of an elution buffer.

The inventive method is carried out, in principle, with the use of the components, described above with respect to the reagent kit. For this reason for a more detailed description of the components, reference is made to the above description. In this respect the addition of a protease is preferred - preferably protease K -, in order to avoid trouble due to the proteins. The particles and the reagents that are used respectively match those described above; similarly the appropriate wash and elution buffers, which are also used in the method, according to the invention. It is especially preferred to use for the washing process at least 60% ethanol. In addition, for the purpose of isolating nucleic acids from samples containing yeast, it is preferred to use a reagent commensurate with reagent I that contains detergent and alcohol. On the other hand, in particular a reagent that comprises chaotropic salts corresponding to reagent II is used for isolating nucleic acids from whole blood.

The method, according to the invention, can be used, in principle, for isolating DNA or RNA. In this case, however, the isolation of DNA is preferred.

An additional subject matter of the present invention is a method for amplifying and/or determining nucleic acids by means of a polymerase chain reaction. In this method the nucleic acid that is to be amplified is isolated from a biological sample, comprising nucleic acid-containing compartments, by means of the above described method of the invention and/or by means of the above described reagent kit of the invention. One specific advantage of the invention lies, in particular, in the fact that after the nucleic acids have been separated from the polymer particles in the elution buffer, the nucleic acids can be added directly to a PCR reaction. The PCR reaction itself and/or the identification of the nucleic acids, for example, by sequencing can be carried out according to well-known methods.

The inventive method and the inventive reagent kit provide simple and easily automatable possibilities for preparing samples for the PCR. Hence, both the method and the kit constitute a significant step towards simplifying even more the analysis of nucleic acid-containing samples.

The following examples shall explain in detail the invention with reference to the figures.

Examples

1. Binding of lambda HindIII marker DNA to magnetic particles, coated with carboxyl groups, as the adsorber and magnetic deposition

In a Thermosprint plate [from the firm Innova, Mannheim] 0.5 mg lambda HindIII marker DNA in 10 ml of double distilled water were treated with a working solution, produced from 250 mg particles composed of polyvinyl alcohol (produced according to EP 0 843 591) in 5 ml of double distilled water, 5 ml of protease K solution [1 373 196, Roche, Mannheim] and 130 ml of BILATEST lysis buffer 1 (SDS) comprising 5% sodium dodecyl sulfate [L4390, Sigma, Munich], 100 mM tris/HCl [T2584, Sigma, Munich], 10 mM EDTA [E5134, Sigma, Munich], 0% up to 80% ethanol [5054.1, Roth, Karlsruhe].

Then the particles are removed with a magnet; and the supernatant is removed; and the particles are washed with 150 ml of 80% ethanol [5054.1, Roth, Karlsruhe] in double distilled water. This procedure is repeated once; thereafter the particles are resuspended in 150 ml of BILATEST elution buffer, comprising 10 mM of tris/HCl having a pH 7.0 [see above] and 1 mM EDTA [see above], incubated for 5 min. at 65 deg. C, and removed, as before, with a magnet. The cleaned nucleic acid is removed in the supernatant. The analysis in a standard agarose flatbed gel (0.8% agarose [A9311, Sigma, Munich]) yields an image, as depicted in Figure 1.

Figure 1

Trace Content

1, 2 eluate from example 1, lysis buffer with 0% ethanol

3, 4 eluate from example 1, lysis buffer with 10% ethanol

Trace Content

5, 6 eluate from example 1, lysis buffer with 20% ethanol

7, 8 eluate from example 1, lysis buffer with 30% ethanol

9, 10 eluate from example 1, lysis buffer with 40% ethanol

11, 12 eluate from example 1, lysis buffer with 50% ethanol

13, 14 eluate from example 1, lysis buffer with 60% ethanol

15, 16 eluate from example 1, lysis buffer with 70% ethanol

17, 18 eluate from example 1, lysis buffer with 80% ethanol

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

The binding of the DNA to the magnetic particles is clearly dependent on the ethanol concentration; below 50% ethanol hardly any binding can be observed.

2. Isolation of nucleic acid from yeast with particles, coated with carboxyl groups, as the adsorber and centrifugation

In a first step a working solution is prepared for a test batch. This working solution is produced from 250 mg of particles composed of polyvinyl alcohol (produced according to EP 0 843 591) in 5 ml of double distilled water, 5 ml of protease K solution [1 373 196, Roche, Mannheim] and 130 ml of BILATEST lysis buffer 1 (SDS) comprising 5% sodium dodecyl sulfate [L4390, Sigma, Munich], 100 mM tris/HCl [T2584, Sigma, Munich], 10 mM EDTA [E5134, Sigma, Munich], 50% ethanol [5054.1, Roth, Karlsruhe].

For a test batch 10⁸ yeast cells (Saccharomyces cerevisiae) in 10 ml volume are treated with 140 ml of this working solution in a Thermosprint plate [Innova, Mannheim] and incubated at 37 deg. C for 15 min. Then the particles are centrifuged in a centrifuge [no. 5810R; Eppendorf, Hamburg] for 2 minutes at 1,000 rpm in a microtitration plate rotor [no. A-4-62; Eppendorf, Hamburg]. The supernatant is removed; and the particles are washed with 150 ml of 80% ethanol [5054.1, Roth, Karlsruhe] in double distilled water. This procedure is repeated once; thereafter the particles are resuspended in 150 ml of BILATEST elution buffer, comprising 10 mM of tris/HCl having a pH 7.0 [see above] and 1 mM EDTA [see above], incubated for 10 min. at 65 deg. C, and removed, as before, by centrifugation. The cleaned nucleic acid is removed in the supernatant. The analysis in a standard agarose flatbed gel (0.8% agarose [A9311, Sigma, Munich]) yields an image, as depicted in Figure 2.

Figure 2

Trace Content

1 eluate from example 2

2 repeat trace 1

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

3. Isolation of nucleic acid from yeast by means of magnetic particles, coated with carboxyl groups, as the adsorber and magnetic deposition

Example 2 was repeated. However, instead of centrifugation, the particles were removed with a permanent magnet [from the firm Rheinmagnet, Neunkirchen].

As a result the eluate was analyzed with a standard agarose flatbed gel (0.8% agarose [see above]) and depicted in Figure 3.

Figure 3

Trace Content

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

1 eluate from example 3

2 repeat trace 1

4. Isolation of nucleic acid from yeast by means of magnetic particles, coated with carboxyl groups, as the adsorber and magnetic deposition

Example 3 was repeated. In this case the elution was not carried out at 65 deg. C, but rather at room temperature.

As a result the eluate was analyzed with a standard agarose flatbed gel (0.8% agarose [see above]) and depicted in Figure 4.

Figure 4

Trace Content

1 eluate from example 4

2 repeat trace 1

Trace Content

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

5. Isolation of nucleic acid from yeast by means of magnetic particles, coated with carboxyl groups, as the adsorber and magnetic deposition

Examples 3 and 4 were repeated for immediate comparison.

As a result the eluate was analyzed with a standard agarose flatbed gel (0.8% agarose [see above]) and depicted in Figure 5.

Figure 5

Trace Content

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

1, 2 eluate from example 5, elution at room temperature

3, 4 eluate from example 5, elution at 65 deg. C

The elution at room temperature leads to a lower elution of smaller nucleic acid molecules, for example, RNA (the bottom two bands in trace 3 and 4). This selectivity can be used to separate a variety of nucleic acid molecules.

6. Isolation of nucleic acid from yeast by means of magnetic particles,

coated with carboxyl groups, as the adsorber and magnetic deposition

Example 3 was repeated. In this case the lysis buffers with SDS or with CTAB (cetyl trimethyl ammonium bromide, 9161.1, Roth, Karlsruhe) were compared, with 50% ethanol or without ethanol respectively.

As a result the eluate was analyzed with a standard agarose flatbed gel (0.8% agarose [see above]) and depicted in Figure 6.

Figure 6

Trace Content

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

1, 2 eluate from example 6, lysis buffer with 5% SDS

3, 4 eluate from example 6, lysis buffer with 5% SDS and 50% ethanol

5, 6 eluate from example 6, lysis buffer with 5% CTAB

7, 8 eluate from example 6, lysis buffer with 5% CTAB and 50% ethanol

CTAB can be used, instead of SDS. However, without ethanol no binding of the genomic DNA takes place in the two cases. The control experiments showed that the cell lysis is achieved even without ethanol. Therefore, the low yield in the samples without ethanol can be traced to the low negligible binding of the DNA in the absence of ethanol.

7. Isolation of nucleic acids from EDTA rabbit whole blood by means of magnetic particles, coated with carboxyl groups, as the adsorber and guanidinium thiocyanate solution

In a first step a working solution is prepared for a test batch. This working solution is produced from 250 mg of particles, composed of polyvinyl alcohol (produced according to EP 0 843 591), in 5 ml of double distilled water, 5 ml of protease K solution [1 373 196, Roche, Mannheim] and 130 ml of BILATEST lysis buffer 1I (Gu-SCN) comprising 6 M of guanidinium thiocyanate (G9277, Sigma, Munich], 100 mM of tris/HCl [T2584, Sigma, Munich], 1 mM EDTA [E5134, Sigma, Munich], 2.5% sodium lauryl sulfate [L9150, Sigma, Munich], pH 7.0.

10 ml of EDTA rabbit whole blood are treated with 140 ml of the above described working solution and incubated in a sealed state at 37 deg. C for 15 min. Then the particles are attracted to the bottom with a permanent magnet; and the supernatant is removed. The particles are washed with 150 ml of 80% ethanol [5054.1, Roth, Karlsruhe] in double distilled water. This procedure is repeated once; thereafter the particles are resuspended in 150 ml of BILATEST elution buffer, comprising 10 mM of tris/HCl having a pH 7.0 [see above] and 1 mM EDTA [see above], incubated for 10 min. at 65 deg. C, and removed, as before; and the cleaned nucleic acid is removed in the supernatant. The analysis in a standard agarose flatbed gel (0.8% agarose [from the firm Sigma, Munich]) yields an image, as depicted in Figure 7.

Figure 7

Trace Content

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

1 eluate from example 7 undiluted

2 repeat trace 1

Trace Content

3 eluate from example 7, diluted 1 : 10

4 repeat trace 3

8. Isolation of nucleic acids from a mixture of EDTA rabbit whole blood and yeast cells by means of magnetic particles, coated with carboxyl groups, as the adsorber

10⁸ yeast cells in 10 ml EDTA rabbit whole blood were treated as in both example 3 and example 7. The analysis in a standard agarose flatbed gel (0.8% agarose [see above]) yields an image, as depicted in Figure 8.

Figure 8

Trace Content

1 eluate from example 8 with the lysis buffer from example 2

2 repeat trace 1

3 eluate from example 8 with the lysis buffer from example 7

4 repeat trace 3

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

This display suggests that the yeast cells can be selectively lysed with the SDS lysis buffer from examples 1 and 2, whereas the whole blood can be selectively lysed with the guanidine buffer from example 4.

9. Isolation of nucleic acids from human EDTA whole blood by means of magnetic particles, coated with carboxyl groups, as the adsorber and guanidinium thiocyanate solution

Example 7 was repeated with human whole blood. The analysis in a standard agarose flatbed gel (0.8% agarose [from the firm Sigma, Munich] yields an image, as depicted in Figure 9.

Figure 9

Trace Content

1 eluate from example 9 with the lysis buffer from example 7

2 repeat trace 1

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

3 eluate from example 9 with the lysis buffer from example 2

4 repeat trace 3

10. Isolation of nucleic acids from human pathogenic yeast Candida albicans by means of magnetic particles, coated with carboxyl groups, as the adsorber

The experiment from example 4 was repeated with 10⁸ cells of human pathogenic yeast Candida albicans. This yeast is distinguished by an especially thick cell wall that is difficult to lyse.

As a result the eluate was analyzed with a standard agarose flatbed gel (0.8% agarose [see above]) and depicted in Figure 10.

Figure 10

Trace Content

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

1 eluate from example 10

2 repeat trace 1

11. Isolation of nucleic acids from the gram positive bacterium Lactococcas lactis by means of magnetic particles, coated with carboxyl groups, as the adsorber

The experiment from example 4 was repeated with the gram positive bacterium Lactococcas lactis. Gram positive bacteria also exhibit an especially thick cell wall that is difficult to lyse. Lysis periods ranging from 1 minute to 15 minutes were used.

As a result the eluate was analyzed with a standard agarose flatbed gel (0.8% agarose [see above]) and depicted in Figure 11.

Figure 11

Trace Content

M marker (lambda HindIII): 23, 119, 416, 614, 412, 312, 010, 56 KB

1, 2 eluate from example 10; lysis 1 min.

3, 4 eluate from example 10; lysis 5 min.

5, 6 eluate from example 10; lysis 10 min.

7, 8 eluate from example 10; lysis 15 min.

The lysis runs very quickly. Just after 1 minute a high DNA yield is obtained. This yield is also in conformity with the observations in our experiments with the yeasts that could also be lysed after just 1 minute.

12. Isolation of nucleic acids from yeast by means of agarose particles, coated with glutamic acid

The experiment from example 4 was repeated with the agarose particles, coated with glutamic acid (G2759, Sigma, Munich). The result was similar to the result of example 4, which is depicted in Figure 4. However, the yield was considerably smaller.

13. PCR Experiments

a) Yeast

The isolated yeast DNA from example 3 was used to run a PCR (US 4 683 195) with the primers

KV80: 5'-GCG GAT CCT TAA GTC CAA TCG TCA AAA TT-3'

KV102: 5'-GCG AAT TCG TAT CTT CTT TGC CCA AGG AA-3'

[from the firm MWG Biotech AG, Ebersberg in the vicinity of Munich]. With these primers a 496 bp fragment from the BCYI gene of the yeast is amplified. The PCR batches contain the following constituents:

0.5 mL primer solution 1 (50 mmol mL^-1 in H₂O double distilled)

0.5 mL primer solution 2 (50 mmol mL^-1 in H₂O double distilled)

2 mL dNTP solution, concentration of the nucleotides per 5 mM (Eurogentec, Seraing, Belgium)

4 mL MgCl₂ solution 25 mM (Eurogentec)

5 mL 10 x PCR buffer (Eurogentec)

0.5 m Taq polymerase (Eurogentec)

up to 30 mL DNA or sample solution

in a total volume of 50 mL. During the preparation the PCR plate is cooled to 4 deg. C. Following addition of the last solution, the samples are mixed once with a pipette. The PCR is carried out in Thermosprint plates [from the firm Innova GmbH, Mannheim] in Primus 96 Plus [MWG Biotech AG, Munich]. The program comprises the following steps:

cover heated to 110 deg. C

3 min. 94 deg. C

27 cycles at

- 30 s 94 deg. C

- 30 s 50 deg. C

- 2 min. 72 deg. C

5 min. 72 deg. C

cooling to 4 deg. C

After the PCR, the samples are treated with 15 - 20% gel loading buffer with EDTA [Sigma, Munich]. The amplificate was analyzed on a standard agarose gel (1.6%) (shown in Figure 12).

Figure 12

Trace Content

1 30 mL DNA solution

2 repeat of trace 1

3 10 ml DNA solution

4 repeat of trace 3

5 1 mL DNA solution

6 repeat of trace 5

7 0.1 mL DNA solution

8 repeat of trace 7

9 0.01 mL DNA solution

10 repeat of trace 9

11 0.001 mL DNA solution

12 repeat of trace 11

a) Human Blood

The isolated human DNA from example 9 is used to run a PCR (US 4 683 195) with the primers

b-Af: 5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3'

b-Ar: 5'CTA GAA GCA TTT GCC GTG GAC GAT GGA GGG-3'

[from the firm MWG Biotech AG, Ebersberg in the vicinity of Munich]. With these primers a 600 bp fragment from the beta-actin gene of the human is amplified.

The PCR batches are carried out as described under a). After the PCR, the samples are treated with 15 - 20% gel loading buffer with EDTA [Sigma, Munich]. The amplificate was analyzed on a standard agarose gel (1.6%) (shown in Figure 13).

Figure 13

Trace Content

1 30 mL DNA solution

2 repeat of trace 1

3 10 mL DNA solution

4 repeat of trace 3

5 1 mL DNA solution

6 repeat of trace 5

7 0.1 mL DNA solution

8 repeat of trace 7

9 0.01 mL DNA solution

10 repeat of trace 9

11 0.001 mL DNA solution

12 repeat of trace 11

M marker 100 bp ladder

Patent Claims

1. Reagent kit for isolating nucleic acids from a biological sample, comprising nucleic acid-containing compartments,

characterized in that it comprises

a) negatively charged particles, composed of a polymer material,

b) a reagent I, comprising a mixture of a charged or uncharged detergent and an aliphatic alcohol, and/or a reagent II, comprising an aqueous solution of at least one chaotropic salt, and

c) a solution of protease.

2. Reagent kit, as claimed in claim 1, characterized in that it contains negatively charged particles composed of polystyrene or, in particular, composed of polyvinyl alcohol.

3. Reagent kit, as claimed in claim 1 or 2, characterized in that the particles are magnetically influencable.

4. Reagent kit, as claimed in any one of the claims 1 to 3, characterized in that the particles exhibit an average diameter ranging from 0.1 to 100 mm and, in particular, from 1 to 10 mm.

5. Reagent kit, as claimed in any one of the preceding claims, characterized in that the particles exhibit carboxyl groups on the surface.

6. Reagent kit, as claimed in any one of the preceding claims, characterized in that the reagent I contains, as the detergent, sodium dodecyl sulfate or DTAB, preferably in an amount ranging from 1 to 10%.

7. Reagent kit, as claimed in any one of the preceding claims, characterized in that the reagent I contains, as the aliphatic alcohol, ethanol, preferably in a concentration of at least 40% by volume.

8. Reagent kit, as claimed in any one of the preceding claims, characterized in that the reagent II contains, as the chaotropic salt, guanidinium hydrochloride or guanidinium thiocyanate, preferably in concentrations ranging from 2 to 8 M.

9. Reagent kit, as claimed in any one of the preceding claims, characterized in that it contains a protease K solution.

10. Reagent kit, as claimed in any one of the preceding claims, characterized in that the reagents I and/or II contain additional buffer and/or complexing agent substances.

11. Reagent kit, as claimed in any one of the preceding claims, characterized in that the reagents I and II exhibit a pH value ranging from 6 to 8.

12. Reagent kit, as claimed in any one of the preceding claims, characterized in that it contains additionally

d) wash buffer and/or

e) elution buffer for releasing the nucleic acids that are bound to the particles.

13. Method for isolating nucleic acids from a biological sample, comprising nucleic acid-containing compartments, characterized in that the sample is treated with negatively charged particles, composed of a polymer material, and with a reagent, comprising a mixture of a charged or uncharged detergent and an aliphatic alcohol, or comprising an aqueous solution of at least one chaotropic salt; then the particles are separated in a suitable way from the supernatant solution, are optionally washed; and then the nucleic acids, bound to the particles, are released from the particles by means of an elution buffer.

14. Method, as claimed in claim 13, characterized in that in addition to the reagent, a protease - preferably protease K - is added.

15. Method, as claimed in claim 13 or 14, characterized in that polystyrene or, in particular, polyvinyl alcohol particles are used as the particles composed of a polymer material.

16. Method, as claimed in any one of the claims 13 to 15, characterized in that magnetically influencable particles are used; and they are separated from the supernatant solution by means of a magnet.

17. Method, as claimed in any one of the claims 13 to 16, characterized in that particles exhibiting an average diameter ranging from 0.1 to 100 mm and, in particular, from 1 to 10 mm, are used.

18. Method, as claimed in any one of the claims 13 to 17, characterized in that particles exhibiting carboxyl groups on the surface are used.

19. Method, as claimed in any one of the claims 13 to 18, characterized in that a reagent, containing sodium dodecyl sulfate or DTAB, preferably in amounts ranging from 1 to 10%, is used for isolating nucleic acids from the samples, containing yeast cells.

20. Method, as claimed in claim 19, characterized in that the reagent uses, as an aliphatic alcohol, ethanol, preferably in a concentration of at least 40% by volume.

21. Method, as claimed in any one of the claims 13 to 18, characterized in that a reagent, containing guanidinium hydrochloride or guanidinium thiocyanate, preferably in concentrations ranging from 2 to 8 M, is used in order to isolate nucleic acids, in particular, from whole blood.

22. Method, as claimed in any one of the claims 13 to 21, characterized in that additional buffer substances and/or complexing agents are added to the reagents.

23. Method, as claimed in any one of the claims 13 to 22, characterized in that the separated, nucleic acid-loaded particles are washed with a wash buffer containing at least 60% ethanol.

24. Method, as claimed in any one of the claims 13 to 23, characterized in that it is carried out using a reagent kit, as claimed in any one of the claims 1 to 12.

25. Method for amplifying and/or determining nucleic acids by means of a polymerase chain reaction (PCR), characterized in that the nucleic acids to be amplified are isolated from a biological sample, containing nucleic acid-containing compartments, by means of a method, as claimed in any one of the claims 13 to 24, and that the amplification reaction as well as optionally the determination are brought about according to well-known methods.

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5 pages of drawings

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- Blank Sheet -

Drawings Page 1 Number: DE 199 37 607 A1

Int. Cl.⁷: C 12 N 15/10

Disclosure date: February 15, 2001

[see figures]