METHOD FOR ENRICHING BIOMOLECULES AND FOR REMOVING THE BIOMOLECULES FROM A BIOLOGICAL SAMPLE

Abstract

A method includes enriching biomolecules and removal of the biomolecules from a biological sample. In the presence of particles, an alginate solution and salts of divalent and/or polyvalent cations or an acid are added to a biological sample, and an alginate-gel-biomolecule-complex is formed on the particles. The complex is removed from the sample by separation of the particles, and from which subsequently the biomolecules or ingredients of the biomolecules are released. The biomolecules, which shall be enriched, include cell-free nucleic acids, viruses or subcellular microparticles. The method is improved and simplified.

Claims

1: A method for enriching a biomolecule and for removal of the biomolecule from a biological sample, said method comprising: adding, in the presence of a particle, an alginate solution and a salt of a divalent cation and/or a polyvalent cation or an acid to the biological sample, wherein an alginate-gel-biomolecule-complex is formed on the particle, removing said complex from the biological sample by separation of the particle, and subsequently releasing the biomolecules or an ingredient of the biomolecule from the alginate-gel-biomolecule-complex.

2: The method according to claim 1, wherein the particle is a magnetic particle or a paramagnetic particle.

3: The method according to claim 1, wherein calcium, zinc, aluminum, or combinations thereof are used as the salt of the divalent cation and/or the polyvalent cation.

4: The method according to claim 1, wherein the biomolecule is selected from the group consisting of cell-free nucleic acids, viruses, subcellular microparticles, and mixtures thereof.

5: The method according to claim 2, wherein the particle is the magnetic particle, separation of the particle from the alginate-gel-biomolecule-complex takes place via a magnet.

6: The method according to claim 1, wherein the release of the biomolecule takes place by dissolving of the alginate-gel-biomolecule-complex, which is located on the particle, wherein the particle and the biomolecule are released.

7: The method according to claim 6, wherein dissolving of the alginate-gel-biomolecule-complex takes place by trisodium citrate dihydrate or by a chelating agent.

8: The method according to claim 6, wherein after dissolving of the alginate-gel-biomolecule-complex located on the particle, resuspending of the particle and an incubation takes place, and the particle is separated.

9: The method according to claim 8, wherein the biomolecule is selected from the group consisting of cell-free nucleic acids, and wherein the particle, on which the alginate-gel-nucleic acid-complex has formed, after dissolving of the alginate-gel-nucleic acid-complex, at the same time binds the cell-free nucleic acid released from the alginate-gel-nucleic acid-complex.

10: The method according to claim 9, wherein after dissolving of the alginate-gel-nucleic acid-complex and resuspension of the particle, a binding buffer for binding the cell-free nucleic acids to the particles is added, and subsequent isolation of the cell-free nucleic acids takes place in a known manner.

11: A kit for carrying out the method according to claim 1, comprising: a) an alginate solution, b) at least one salt of a divalent cation and/or a polyvalent cation or an acid, c) a particle, d) a separator of the particle which has formed a complex from a) and b) the biomolecule, and e) means for releasing the of biomolecule which is bound in a complex of a), b), and c).

12: The method according to claim 7, wherein the chelating agent is EDTA.

13: The kit according to claim 11, wherein e) is a chelating agent.

14: The method according to claim 1, wherein the biological sample is at least one member selected from the group consisting of serum, plasma, and urine.

Description

EMBODIMENT 1

[0031] Enrichment of cell-free DNA from a plasma sample of 1 ml. Proof of the necessary combination of alginate solution, reagent for formation of an alginate gel as well as particles separable by means of a magnetic field

[0032] Initial sample for the enrichment had been human plasma. The plasma sample had been centrifuged again for 10 minutes prior to the application in order to remove still existing cells, if applicable. Further processing took place with the supernatant. Three different processes have been tested.

[0033] Sample 1: 30 l of a 0.5% alginate solution were added to the sample and the batch was briefly mixed. Subsequently, 150 l of a 1 molar calcium chloride solution were added as well as 50 l of a magnetic particle suspension (MAG Suspension; Analytik Jena AG).

[0034] Sample 2: 30 l of a 0.5% alginate solution were added to the sample and the batch was briefly mixed. Subsequently 50 l of a magnetic particle suspension (MAG Suspension; Analytik Jena AG) were added.

[0035] Sample 3: 150 of a 1 molar calcium chloride solution were added to the sample and the batch was briefly mixed. Subsequently 50 l of a magnetic particle suspension (MAG Suspension; Analytik Jena AG) were added.

[0036] The samples were then processed further as follows.

[0037] The batch was briefly mixed and incubated for 10 minutes. Subsequently, separation of the magnetic particles by means of a magnet took place. The supernatant was removed and the magnetic particles were washed with 1 ml water. After renewed separation of the magnetic particles, the supernatant was completely removed.

[0038] 400 l buffer (4 M guanidine thiocyanate, EDTA) as well as 20 l proteinase K (20 mg/ml) were added to the magnetic particle-alginate-gel-DNA-complex and the batch was resuspended by means of a pipette. Subsequently, an incubation for 15 minutes at 70 C. took place.

[0039] This step served for destruction of the particle-alginate-gel-DNA-complex and thus the release of the complexed DNA. Subsequently, the addition of 400 l of a binding buffer (isopropanol/triton X-100) and a renewed mixing of the particles as well as an incubation for 2 minutes took place. This step served for binding of the DNA to the particles. Subsequently, the magnetic particles were separated and the supernatant was discarded. Thereafter, the particles were washed with alcoholic wash buffers known to the person skilled in the art, and finally dried. In the last step, the bound DNA was detached from the particles by the addition of 50 l H.sub.2O, and transferred to a new reaction vessel.

[0040] The proof of enrichment and subsequent extraction of cell-free DNA took place by means of Real Time PCR. For this purpose, a human specific target sequence oestrogen receptor 1 has been amplified.

Protocol RealTime PCR for Amplification of the human specific Target Sequence (Oestrogen Receptor 1).

TABLE-US-00001 sensePrimer (5-CGCCGCCAACGCGCAGGTCTA-3) antisensePrimer (5-AGCCGAACGCCGCAGCCTCA-3) 1probe (5-FAMCCTCCCCTACGGCCCCGGG-BHQ1-3) [0041] Reaction mixture [0042] per sample:

TABLE-US-00002 sense Primer (50 pmol/l) 0.1 l antisense Primer (50 pmol/l) 0.1 l probe (25 pmol/l) 0.1 l dNTP-Mix (12.5 mM) 0.3 l 10X PCR buffer (MgCl.sub.2 included) 1.5 l Taq-DNA-Polymerase 0.75 U PCR-Grade H.sub.2O add 15 l [0043] Amplification/Hybridization Conditions

TABLE-US-00003 Step 1: Denaturation 95 C. 120 Step 2 Amplification 45 cycles 95 C. 4 (measurement) 65 C. 45

Result PCR

[0044] The results in FIG. 1 show that only the combination of alginate solution, reagent for the formation of an alginate gel as well as particles separable by means of a magnet field permits an enrichment and subsequent extraction of DNA (black graphs). Thus, it is proven that the first step is the complexation of free DNA with the forming alginate gel, and that the added particles combine with this complex. Thus, by applying a magnet field, the particle-alginate-gel-DNA-complex can be separated. Hence, a centrifugation, as this is described in the patent specification DE 10 2008 023 297 B4, is not necessary.

EMBODIMENT 2

[0045] Enrichment of cell-free DNA from a plasma sample of 1 ml as well as of 5 ml and subsequent extraction of DNA

[0046] Human plasma had been the initial sample for enrichment. Prior to application, the plasma sample had been centrifuged again for 10 minutes in order to remove still existing cells, where applicable. Further processing took place with the supernatant.

[0047] The 1 ml sample has been treated as follows. 30 l of a 0.5% alginate solution were added to the sample, and the batch was briefly mixed. Subsequently, 150 l of a 1 molar calcium chloride solution were added as well as 50 l of a magnetic particle suspension (MAG Suspension; Analytik Jena AG). The batch was briefly mixed and incubated for 10 minutes. Subsequently, separation of the magnetic particles by means of a magnet took place. The supernatant was removed, and the magnetic particles were washed with 1 ml water. After renewed separation of the magnetic particles, the supernatant was completely removed.

[0048] 400 l buffer (4 M guanidine thiocyanate, EDTA) as well as 20 l proteinase K (20 mg/ml) were added to the magnetic particle-alginate-gel-DNA-complex, and the batch was resuspended by means of a pipette. Subsequently, an incubation for 15 minutes at 70 C. took place.

[0049] This step served for destruction of the particle-alginate-gel-DNA-complex, and thus the release of the complexed DNA. Subsequently, the addition of 400 l of a binding buffer (isopropanol/triton X-100) and a renewed mixing of the particles as well as an incubation for 2 minutes took place. This step served for binding of the DNA to the particles. Subsequently, the magnetic particles were separated, and the supernatant was discarded. Thereafter, the particles were washed with alcoholic wash buffers known to the person skilled in the art, and finally dried. In the last step, the bound DNA was detached from the particles by the addition of 50 l H.sub.2O, and transferred to a new reaction vessel.

[0050] The 5 ml sample were treated as follows. The sample was transferred to a 15 ml reaction vessel. 150 l of an 0.5% alginate solution were added to the sample and the batch was briefly mixed. Subsequently 600 l of a 1 molar calcium chloride solution as well as 100 l of a magnetic particle suspension (MAG Suspension; Analytik Jena AG) were added. The batch was briefly mixed and incubated for 10 minutes. Subsequently, separation of the magnetic particles by means of a magnet took place. The supernatant was removed and the magnetic particles were washed with 5 ml water. After renewed suspension of the magnetic particles, the supernatant was completely removed.

[0051] 400 l buffer (4 M guanidine thiocyanate, EDTA) as well as 20 l proteinase K (20 mg/ml) were added to the magnetic particle-alginate-gel-DNA-complex, resuspended, and the batch was transferred by means of a pipette into an 1.5 ml reaction vessel. Subsequently, an incubation for 15 minutes at 70 C. took place.

[0052] This step served for destruction of the particle-alginate-gel-DNA-complex and thus the release of the complexed DNA. Subsequently, the addition of 400 l of a binding buffer (isopropanol/triton X-100) and a renewed mixing of the particles as well as an incubation for 2 minutes took place. This step served for binding of the DNA to the particles. Subsequently, the magnetic particles were separated and the supernatant was discarded. Thereafter, the particles were washed with alcoholic wash buffers known to the person skilled in the art, and finally dried. In the last step, the bound DNA was detached from the particles by the addition of 50 l H.sub.2O, and transferred to a new reaction vessel.

[0053] The proof of enrichment and subsequent extraction of cell-free DNA took place by means of Real Time PCR. For this purpose, a human specific target sequence (oestrogen receptor 1) has been amplified.

Results Real Time PCR

[0054] Protocol Real Time PCR for amplification of human specific target sequence (oestrogen receptor 1). Protocol see example 1.

TABLE-US-00004 Sample Ct values 1 ml sample (red graph) 35/34.95 5 ml sample (black graph ) 32.25/32.30

[0055] As the results in FIG. 2 show, from the 5 ml sample more DNA is enriched and subsequently isolated than from the 1 ml sample. Thus, the method according to the invention is suitable also for the enrichment and extraction cell-free of DNA from large volume samples.

EMBODIMENT 3

[0056] Enrichment of genomic DNA from an aqueous solution (1 ml) and direct release of the genomic DNA without further DNA extraction. Comparison of the method according to the invention with the method from the patent specification DE 10 2008 023 297 B4

[0057] The initial sample for enrichment was an aqueous solution which contained genomic DNA. Enrichment occurred by means of the method from the patent specification DE 10 2008 023 297 B4 as well as by means of the method according to the invention without a centrifugation step. For the method from the patent specification, the commercial product PME free-circulating DNA Extraction Kit (Analytik Jena AG) has been used. The method according to the invention was carried out as follows.

[0058] 30 l of a 0.5% alginate solution were added to the sample, and the batch was briefly mixed. Subsequently, 150 l of a 1 molar calcium chloride solution were added as well as 50 l of a magnetic particle suspension (MAG Suspension; Analytik Jena AG). The batch was briefly mixed and incubated for 5 minutes. Subsequently, separation of the magnetic particles by means of a magnet took place. The supernatant was removed, and the magnetic particles were washed with 1 ml water. After renewed separation of the magnetic particles, the supernatant was completely removed. The magnetic particle-alginate-gel-DNA-complex was subsequently destroyed in order to release the DNA. For this purpose, 50 l of a 50 mM sodium citrate solution were added to the particles, and the batch was resuspended by means of a pipette. After a short incubation, the magnetic particles were separated, and the supernatant was transferred to a new vessel, and subsequently 50 l water were added. The analysis as to whether the genomic DNA of the sample was enriched also without centrifugation, took place on an agarose gel. As it is shown by the gel-electrophoretic representation, the genomic DNA from the 1 ml sample could be concentrated by means of both methods. In this context, the method according to the invention distinguishes itself by its simplicity because no more centrifugation steps were required.

[0059] FIG. 3 shows the gel-electrophoretic representation of the concentrated DNA by means of both methods, wherein No. 1 shows the 1-DNA ladder, Nos. 2 to 5 show the concentrated genomic DNA by means of the method from patent specification DE 10 2008 023 297 and Nos. 6 to 9 show the concentrated genomic DNA by means of the method according to the invention.

EMBODIMENT 4

[0060] Enrichment of Viruses and subsequent Extraction of the viral Nucleic Acid

[0061] Human plasma was the initial sample for the enrichment. Inactivated yellow fever virus was added to the plasma sample. For enrichment of the viruses, 1 ml sample was used. The 1 ml sample was treated as follows. 30 l of a 0.5% alginate solution were added to the sample, and the batch was briefly mixed. Subsequently, 150 l of a 1 molar calcium chloride solution were added as well as 50 l of a magnetic particle suspension (MAG Suspension; Analytik Jena AG). The batch was briefly mixed, and incubated for 10 minutes. Subsequently, separation of the magnetic particles by means of a magnet took place. The supernatant was removed, and the magnetic particles were washed with 1 ml water. After renewed separation of the magnetic particles, the supernatant was completely removed.

[0062] 400 l buffer (4 M guanidine thiocyanate, EDTA) as well as 20 l proteinase K (20 mg/ml) were added to the magnetic particle-alginate-gel-DNA-complex, and the batch was resuspended by means of a pipette. Subsequently, an incubation for 15 minutes at 60 C. took place.

[0063] This step served for destruction of the particle-alginate-gel-virus-complex and thus the release of the complexed viruses and destruction for release of the viral nucleic acid (viral RNA). Subsequently, the addition of 400 l of a binding buffer (guanidine thiocyanate/isopropanol/triton X-100) and a renewed mixing of the particles as well as an incubation for 2 minutes took place. This step served for binding of the viral RNA to the particles. Subsequently, the magnetic particles were separated and the supernatant was discarded. Thereafter, the particles were washed with alcoholic wash buffers known to the person skilled in the art, and finally dried. In the last step, the bound DNA was detached from the particles by the addition of 50 l H.sub.2O, and transferred to a new reaction vessel.

[0064] The proof of enrichment of the virus particles, and the subsequent extraction of the viral RNA took place by means of a yellow fever virus specific Real Time PCR.

Results Real Time PCR

[0065] Protocol Real Time PCR for amplification of the 5 Noncoding Region of Yellow Fever Virus

TABLE-US-00005 YcaseF: (5-GCTAATTGAGGTGYATTGGTCTGC-3) YcaseR (5-CTGCTAATCGCTCAAMGAACG-3) YcaseP (5-FAM-ATCGAGTTGCTAGGCAATAAACAC-BHQ1-3)
Reaction batch [0066] per sample:

TABLE-US-00006 Y case F (50 pmol/l) 0.1 l Y case R (50 pmol/l) 0.1 l Y case P (25 pmol/l) 0.1 l dNTP-Mix (12.5 mM) 0,3 l 10X PCR buffer (MgCl.sub.2 included) 1.5 l Taq-DNA-Polymerase 0.75 U PCR-Grade H.sub.2O add up to 15 l [0067] Amplification/Hybridization conditions

TABLE-US-00007 Step 1: Denaturation 95 C. 120 sec Step 2 Amplification 45 cycles 95 C. 4 sec (measurement) 65 C. 45 sec

Results PCR

[0068]

TABLE-US-00008 Sample Ct values 1 ml sample (blue) 28.8/28.9 [0069] In FIG. 4 it has been shown that also viruses are enriched, and subsequently the viral nucleic acid can be extracted.