DE102005031910A1 - Method for stabilizing or isolating nucleic acids - Google Patents

Abstract

The present invention provides a method for stabilizing or isolating nucleic acids in a biological sample with amine surfactant compounds. The mechanisms of nucleic acid stabilization or isolation of the present invention are different from conventional methods. Surfactants with primary amines, secondary amines and tertiary amines, or the mixtures with various ratios of surfactants are used in the present invention to stabilize or isolate nucleic acids by forming insoluble ionic complexes between nucleic acids and a surfactant. The method of the present invention may even be performed automatically to increase throughput and to extend the application of molecular diagnostic testing with nucleic acid.

Description

BACKGROUND OF THE INVENTION

1st area the invention

The The present invention relates to a method for stabilizing or Isolating biomaterials from a sample and in particular a Method for stabilizing or isolating nucleic acids a biological sample.

2. Description of the subject

It is known to be nucleic acids carry the genetic information of an organism. Play nowadays nucleic acids also an important role in research areas of molecular biology. According to recent research results It is known that genetic defects or the development of Diseases of a patient by clinical practice from the abnormality or special Sequences of nucleic acids of this Patients can be derived. Consequently, the goal for preventing the occurrence of a disease before the onset of the disease by detecting the abnormality of nucleic acids and performing necessary remedial steps for treatment are achieved. To obtain an effective proof of the abnormality or special Sequences of the nucleic acids it is the isolation of nucleic acids from an organism and the steps of keeping the genetic information intact around key tasks for those concerned Applications.

Nucleic acids are active molecules, especially RNA. conventional Methods for isolating active RNA molecules are generally associated with Anticoagulants, e.g. EDTA in the phlebotomized whole blood applied, after which the sample at 4 ° C is stored until the isolation steps can be performed. The RNA expression levels would by the addition of anticoagulants, temperature changes, the storage times and the isolation process for leucocytes are affected, these factors are the difficulties of predicting the occurrence a disease by RNA expression increase. To the Obtaining better results must be the isolation of RNA from whole blood samples be carried out within 24 hours. However, that means this mandatory processing time usually, especially if big Sample quantities arrive for processing, an unacceptable burden for the medical professionals.

To the Avoid contamination of proteins and other organelles These are the steps of isolating pure nucleic acids below Involvement of the application of phenol / chloroform in conventional Procedure common. However, the use of phenol / chloroform in the steps requires isolating nucleic acids from biological materials a careful way of working and careful technical staff, around unwanted Exclude organelles or materials (e.g., proteins) from the nucleic acids. Farther are phenol and chloroform for the process dangerous and heavily polluting. In addition, phenol and chloroform nucleic acids do not condense directly. That is why many chemicals are there to help Phenols or chloroforms when condensing nucleic acids needed. There are also phenols inconvenient to store and use as they oxidize easily. According to these The disadvantages described above are phenol / chloroform not the best chemical for use in isolating nucleic acids it especially for high throughput isolation of nucleic acids for clinical biomedical Applications is unsuitable.

Some Studies on the isolation of nucleic acids without phenol / chloroform were discussed. Qiagen Company disclosed in PCT Patent No. WO03084976 for coping with disadvantages illustrated above, a method of isolation of nucleic acids with ammonia or ammonium. In this procedure, a fixed Phase, which acts as an attachment substrate for binding nucleic acids. additionally At the beginning of the isolation stage, a chaotropic agent is present to melt double-stranded oligonucleotides. In addition, in the presence of the Ammonia or the primary Amines the binding efficiency between denatured nucleic acids and the solid phase improved. Accordingly, it is preferable that the Contacting the sample with the nucleic acid-binding solid phase in the presence of ammonium or ammonia at a pH range from 8.5 to 9.5 becomes.

US 6,265,168 to Gjerde et al. discloses a method for removing a target DNA fragment with a predetermined base pair length of a mixture of DNA fragments. The mixture of DNA fragments containing the target DNA fragment is present in a first solvent mixture having a counter ion and a DNA binding concentration of the transport solvent and then applied to a separation column containing a medium having a non-polar, nonporous surface. The target DNA fragments are separated from the medium by contacting the surfaces with a second solvent solution. The counterion used herein are primary amines, secondary amines, lower tertiary amines, and alkyl quaternary ammonium salts. In the key factors of the procedure in US 6,265,168 Purification of a target DNA fragment is the nonpolar and nonporous surface column and the counterion DNA complexes that are readily absorbed on the surface. This disclosure provides a method of isolating target DNA fragments from a purified DNA pool. However, the use of purifying nucleic acids directly from blood is not yet disclosed in the art because of the potential column clogging.

In WO 2004013155 discloses Goldsborough et al. a method for stabilizing of nucleic acids in a biological sample. In the main steps to stabilize of nucleic acids First, this modification is modifying of 2'-, 3'-, and 5'-OH groups one nucleic acid with a protecting group, which helps prevent nucleic acid digestion by nuclease, after which the modified nucleic acids for Remove the protecting group with primary amines. The primary used here Amin serves more for cleavage of the protecting group than for forming a complex with nucleic acids.

Also revealed US 20040048384 to Augello, Frank A. et al. a collection container and a method for collecting a predetermined volume of a biological sample, wherein the whole blood sample includes at least one gene induction blocking agent in an amount effective to stabilize and inhibit gene induction. The stabilizing agent of the gene induction blocking agent disclosed herein is a quaternary amine. A more detailed discussion of the subject method will be apparent from the description of CA 2299119, which discloses a method for stabilizing and / or isolating nucleic acids. The method described here uses at least two quaternary amines or cationic polymers with a phosphorus group to precipitate and protect nucleic acids.

In addition, a novel composition for isolating and / or stabilizing nucleic acids from biological materials in US 2004014703 disclosed. Abandonment of the procedure of US 2004014703 is the provision of a composition for stabilizing RNA in the presence of a tissue, blood, plasma or serum. The composition comprises a cationic compound such as a quaternary amine for stabilizing nucleic acids.

If also the two products "PAXgene Blood RNA Tube "and" PAXgene Blood RNA Isolation Kit " and were commercialized by Qiagen Company and the two Products for stabilizing and isolating nucleic acids in Working together with whole blood. However, the kits are not cost effective, which means the applications of the kit complicate routine uses.

It gives no revelation stabilizing or isolating nucleic acids with primary amine, secondary amine and tertiary Amine. That is why it is desirable an improved method of mitigating and / or circumventing the above mentioned To provide problems.

SUMMARY THE INVENTION

The The present invention provides a method for stabilizing or Isolate nucleic acids in a biological sample with amine surface-active compounds ready. The mechanisms of nucleic acid stabilization or isolation of the present invention are different from conventional ones Method. surfactants Agent with primary Amines, secondary Amines and tertiary Amines or mixtures with different ratios of surface-active Agents are used in the present invention for stabilizing or Isolate nucleic acids by making insoluble ones ionic complexes between nucleic acids and a surface-active Means used. For example, the complexes protect RNA within, to a RNA degradation by RNase as well as to prevent an RNA transcription, whereby the nucleic acids be stabilized and protected in the biological sample and the RNA also is easy to isolate with the complexes.

The Method of the present invention may even be performed automatically to increase throughput and to expand the application of molecular diagnostic testing with nucleic acid.

To achieve the object, the reagent and method of the present invention for stabilizing or isolating nucleic acids by imaging an insoluble ionic complex between nucleic acids and a surfactant in a sample comprises a step of contacting the biological sample with a stabilizing or isolating reagent the surface active amine compound stabilizing or insulating reagent of the formula (I) consists of: R ₁ R ₂ R ₃ N (O) _x , (I) wherein R ₁ and R _{2 are} each independently H, a C 1 -C 6 alkyl group, C 6 -C 12 aryl group or C 6 -C 12 aralkyl group; R _{3 is} a C 1 -C 20 alkyl group, C 6 -C 26 aryl group or C 6 -C 26 aralkyl group; and x is an integer of 0 or 1. One of the best embodiments of the present invention is when in the above formula (I), R ₁ and R _{2 are} each independently H or a C ₁ -C 6 alkyl group, and R _{3 is} a C 1 -C 20 alkyl group when x is 0.

at the surface active Amine compound of the present invention may be any of conventional surfactants Amine compound act. Preferably, the surface-active Amine compound of the present invention selected from the group consisting from dodecylamine, N-methyldodecylamine, N, N-dimethyldodecylamine, N, N-dimethyldodecylamine N-oxide and 4-tetradecylaniline.

The Stabilizing or insulating agent may be in solution or be added as a solid. The option of adding as a solid includes the additional Advantages that solids mostly a higher chemical stability often easier to add to the sample can. Consequently, the mode of contact of the biological sample and the Stabilizing or isolating agent of the present invention not limited this is a liquid solution or a solid can act. However, it is in the Reagent of the preferred embodiment of the present invention for obtaining a better mixing result the sample and the reagent around a liquid solution.

Becomes the reagent added as a solid is the weight percent the surface active Amine compounds in the reagent is not limited and is preferably less than 90% and is stronger preferably in the range of 10 to 90%. Will the reagent be in solution form added, the concentration of the amine surfactant compounds in the reagent solution preferably in the range of 0.001 to 20%.

The Process of the present invention can be used without any presence be carried out by non-ionic detergents or acids. However, could the use of non-ionic detergents, acids or the mixture thereof with the participation of the specific surface-active Amine compounds the stabilization or isolation results improve. Consequently, the stabilizing or isolating reagent with surface active Amine compounds further selectively at least one nonionic detergent include. The at least one nonionic detergent may be present in the stabilizing or isolation reagent as a liquid Detergent or as a solid detergent.

The Concentration of the at least one nonionic detergent is preferably in the range of 0.01 to 20% when the stabilizer or isolation reagent in liquid Condition exists; the weight percentage of at least one Detergent ranges from 0.01 to 40% when the reagent is in the solid state. The at least one nonionic detergent The present invention may be any; preferably is the at least one nonionic detergent polyoxyethylene sorbitan monolaurate. Stronger preferred is the non-ionic detergent Tween 20 or Triton X-100.

The Stabilizing or insulating reagent with surface active Amine compounds of the present invention may further be selective at least one acid include. The at least one acid may be acid buffer or acid to act in a solid state. The concentration of at least one acid buffer is less than 1 M. Preferably, the concentration of at least an acid buffer in the range of 0.01 to 0.5 M. The at least one acid can be any. More preferred is the at least one acid selected from the group consisting of maleic acid, tartaric acid, citric acid, oxalic acid, carboxylic acids and Mineral acids. The pH of the stabilizing or isolating agent of the present invention Invention may be any value in the range of 1 to 14. Preferably, the pH is in the range of 1 to 10.

at the biological sample used in the present invention with nucleic acids it can be a cell-free sample, plasma, body fluid such as blood, serum, cells, leukocyte fractions, sputum, urine, semen, faeces, Swabs, aspirates, tissue samples of all kinds such as biopsies e.g. Parts of tissues and organs, food samples, free or bound nucleic acids or cells containing nucleic acids as provided according to the invention like organisms (single or multicellular organisms, insects, etc.), Plants or parts of plants, bacteria, viruses, yeasts and others Fungi, other eukaryotes and prokaryotes etc. act.

Of the Term "Nucleic Acids" for purposes of reference the present invention nucleic acids in a broader sense and includes hence e.g. ribonucleic acids (RNA) and also deoxyribonucleic acids (DNA) in all lengths and configurations like duplex, single strand, circular and linear, branched, etc., and all possible subunits thereof such as monomeric nucleotides, oligomers, plasmids, viral and bacterial DNA and RNA as well as genomic and non-genomic DNA and RNA from Animal and plant cells or other eukaryotes, mRNA in processed and unprocessed form, tRNA, hn-RNA, rRNA, cDNA, and others imageable nucleic acids one. Preferably, the nucleic acids of the present invention DNA or RNA.

Other Objects, advantages and novel features of the invention will become apparent from the following detailed description clearer when in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

1 is an electrophoresis result of purified RNA in Examples 1-3 of the present invention;

2 is an electrophoresis result of purified RNA in Examples 4-6 of the present invention;

3 Figure 4 is a quantitative RT-PCR result of purified RNA in Examples 7-9 of the present invention showing the relative levels of gene expression in various conservation periods;

4 is an electrophoresis result of purified nucleic acids with dodecylamine from whole blood;

5 is an electrophoresis result of purified nucleic acids with N, N-dimethyldodecylamine from whole blood;

6 shows electrophoresis results of purified nucleic acids with N, N-dimethyldodecylamine N-oxide from whole blood; and

7 shows a result of electrophoresis isolated from purified nucleic acids with 4-tetradecylaniline.

DETAILED DESCRIPTION THE PREFERRED EMBODIMENT

The advantages of the present invention are illustrated by the following examples and figures. Embodiments of stabilizing nucleic acids in a biological sample of the stabilizing or isolating reagent are described in Examples 1-11 and following 1 - 3 described. Also, Examples 12 to 18 and 4 - 7 for the preferred embodiments of isolating nucleic acids in a biological sample.

Stabilize of nucleic acids

Of the RNA expression level is extracted by the amount and quality of the RNA out for a period of several days through three different preservation methods preserved whole blood samples to evaluate stabilization efficiency of nucleic acids certainly.

example 1

One Blood collection tube with a volume of 10 ml of the type Vacutainer (EDTA K3, Becton Dickinson) is used to collect the whole blood samples. The sample in Vacutainer is at 4 ° C for one Stored for 0 to 4 days and then the RNA after the storage period isolated.

According to the manual 1 ml Red Blood Cell Lysis Buffer (Roche Diagnostics GmbH) 500 μl whole blood added for the purification of leukocytes; then 150 μl of buffer RLT (Qiagen GmbH) added to the leukocytes for cell lysis; 90 μl ethanol will be afterwards added to the sample. The sample is then placed in a centrifuge tube (Qiagen GmbH) with silica membrane, and a centrifugation step is carried out.

The Silica membrane in the centrifuge tube is with 350 ul Buffer RW1 (Qiagen GmbH), and the DNA molecules on the Filters become with RNase-free DNase Set (Qiagen GmbH) removed. The silica membrane then becomes again with 350 μl Buffer RW1 and twice with 500 μl Buffer RPE (Qiagen GmbH). The RNA molecules on the Finally, silica filters are washed twice with 40 μl RNase-free Water elutes.

Example 2

in the present example is the collection of whole blood and RNA extraction with PAXgene Blood RNA Validation Kit (Qiagen GmbH). The blood samples will be at 4 ° C for one Duration of 1 to 4 days refrigerated and some of them edited immediately without cooling. After different Storage is RNA according to the in Manual as recommended by the supplier recommended procedure directly extracted.

Example 3

in the The present example is the whole blood sample for RNA extraction with N-methyldodecylamine stored to stabilize RNA.

333 μl fresh Whole blood is mixed with 1 ml of a stabilizing solution consisting of 3% (w / v) N-methyldodecylamine, 5% (v / v) Triton X-100 and 100 mM tartaric acid mixed and for a period of 0 to 4 days at 4 ° C cooled. To isolate the RNA become complexes of surface-active secondary Amine compound and RNA at 5000 xg for 10 minutes centrifuged. The pellet is dissolved in 50 μl of distilled water. 100 μl buffer RLT (QIAGEN GmbH) and 10 μl Proteinase K (QIAGEN GmbH) are added to the sample and kept at 55 ° C for a period of time incubated for 10 minutes. 200 μl 1-Bromo-3-chloropropane is added to the sample and swirled mixed. The sample is then used for centrifuged for 5 minutes at 10,000 xg.

The supernatant will then be in a new tube transferred with a volume of 1.5 ml. The Sample is mixed with 90 ml of ethanol and then loaded onto a spin column a silica membrane applied. The sample mixture is centrifuged passed through the membrane. The silica membrane is filled with 350 μl buffer RW1 (QIAGEN GmbH), and the DNA molecules are used of the RNase-free DNase Set (QIAGEN GmbH). The silica membrane is mixed with another aliquot of 350 μl buffer RW1 and twice with 500 μl buffer RPE (QIAGEN GmbH). Finally, the RNA molecules become twice with the same 40 μl aliquot RNase-free water elutes.

Example 4

in the The present example is the whole blood sample for RNA extraction stored with dodecylamine to stabilize RNA. A stabilizing solution, consisting from 0.3% (w / v) dodecylamine, 1% (v / v) Triton X-100 and 250 mM tartaric acid prepared and the pH adjusted to pH 3 with NaOH. 1 ml fresh whole blood is mixed with 3 ml of the stabilizing solution and then the sample for stored for a period of 0 to 4 days at 4 ° C.

To isolate the RNA, the complexes of the amine surfactant compound and the RNA are collected by centrifugation. The pellet is dissolved in 150 μl of distilled water. 300 μl of buffer RLT (QIAGEN GmbH) and 30 μl of proteinase K (QIAGEN GmbH) are added to the sample and incubated at 55 ° C. for a period of 10 minutes. 200μ 1-bromo-3-chloropropane is added to the sample and mixed by vortexing. The sample is then centrifuged for 5 minutes at 10,000 xg. The supernatant will then transferred to a new tube with a volume of 1.5 ml. The sample is mixed with 270 μl of ethanol and then applied to a spin column containing a silica membrane.

Subsequently, will proceed as in Example 3.

Example 5

in the The present example is the whole blood sample for RNA extraction with N, N-dimethyldodecylamine stored to stabilize RNA. A stabilizing solution, consisting from 5% (w / v) N, N-dimethyldodecylamine, 2% (v / v) Triton X-100 and 140 mM tartaric acid produced. 333 μl fresh whole blood are mixed with 1 ml stabilizing solution, and the mixture is for frozen for a period of 0 to 14 days at -20 ° C. The proceedings of RNA isolation are performed as described in Example 3.

Example 6

in the The present example is the whole blood sample for RNA extraction with a stabilizing solution, consisting of 3% (w / v) N, N-dimethyldodecylamine N-oxide, 1% (v / v) Triton X-100 and 125 mM tartaric acid, stored. The sample mixture is kept for a period of 0 to 14 days stored at -20 ° C. RNA in the samples of different storage periods is determined according to the in Example 3 described isolated.

Example 7

One Agilent 2100 Bioanalyzer (Agilent Technologies) is being analyzed of 28S / 18S rRNA ratios of RNA isolated in Examples 1-3. According to the of Professional recommended standard ratio indicates the 28S / 18S rRNA ratio of higher than 1.5 indicates that the RNA molecules are intact; continue the isolated RNA molecules in good condition when the 28S / 18S rRNA ratio is around 2.0. Also shows a good quality the RNA sample has an OD260 / 280 ratio in the range of 1.9-2.1, determined by a spectrophotometer becomes. The methods described above who the to analyze the quality and amount of RNA samples used, and the results are shown below listed Table 1 listed.

Table 1

Seemingly The results of Example 3 in Table 1 show a higher RNA yield than Example 1 or Example 2. A mean RNA yield of 7.20 ± 0.48 μg can be obtained with the stabilizing solution of the present invention can be isolated in Example 3. Also, a ratio of OD 260/280 1.98 ± 0.14 and the 28S / 18S rRNA ratio 1,830.17, both being the highest quality value of 2.0.

1 shows electrophoresis results of purified RNA in Examples 1-3, wherein (a) is RNA obtained from Example 1; (b) was obtained from Example 2 and (c) was obtained from Example 3. The numbers 0-4 represent the storage days. In the electrophoresis results, the first band of each lane represents 28S rRNA and the second band represents 18S rRNA. Obviously, RNA isolated by the method of the present invention in Example 3 has the Best quality and quantity compared to the other two methods in Example 1 or 2 on. The quantity and the quality show no differences between the RNA samples from the 4-day storage and from the fresh blood (0-day storage).

2 shows electrophoresis results of purified RNA in Examples 4-6, wherein (a) the RNA of Example 4 was obtained with 0-2 days storage; (b) was obtained from Example 5 with storage for 0-14 days and (c) obtained from Example 6 with 0-14-day storage. Data from the Agilent 2100 Bioanalyzer (Agilent Technologies) are listed in Table 2 below. According to the results of Table 2 and 2 shows RNA isolated from whole blood even after 14 days of storage an acceptable condition.

Example 8

All Procedures in the example will be as in the description in Example 1 performed, the whole blood for a duration of 0-2 days at 4 ° C is stored.

Example 9

All Procedures in the example will be as in the description in Example 2 performed, the whole blood for a duration of 0-2 days at 4 ° C is stored.

Example 10

in the present example is 1 ml of whole blood sample for RNA extraction with 3 ml of a stabilizing solution, consisting of 5% (w / v) N, N-dimethyldodecylamine and 225 mM tartaric acid (the pH is adjusted to pH 3.0 with NaOH). The sample mixture is for a duration of 0-2 days at 4 ° C stored. The methods of RNA isolation become different Storage times as described in Example 4 performed.

Example 11

Single-stranded cDNA molecules are synthesized with the RNA molecules isolated from Example 8-10 by SuperScript II RNase H Reverse Transcriptase (Invitrogen) according to the procedures recommended in the manual as recommended by the supplier. The synthesized single-stranded cDNA molecules are then performed with TaqMan Universal PCR Master Mix (Applied Biosystems) and Assays-on-Demand Gene Expression Products (Applied Biosystems), followed by a real-time PCR procedure using the ABI Prism 7000 Sequence Detection System ( Applied Biosystems). The expression levels of the 4 genes ADORA2A, CREB5, NFKB1 and IFNGR1 are determined in different storage periods according to the methods described above, and the results are in 3 shown.

In 3 For example, the relative expression fold values represent the ratio of gene expression levels in RNA isolated from the samples stored at 4 ° C over a period of 24 hours or 48 hours as compared to no storage. For example, the relative expression folding value of 1 represents that the degree of gene expression of the isolated RNA molecules after a certain period of storage is the same as that of a 0-hour storage. A1-A4 in 3 are the results of Example 8, wherein A1 is the gene ADORA2A, A2 is the gene CREB5, A3 is the gene IFNGR1 and A4 is the gene NFKB1. B1-B4 in 3 are the results of Example 9, where B1 is the gene ADORA2A, B2 is the gene CREB5, B3 is the gene IFNGR1 and B4 is the gene NFKB1. C1-C4 in 3 are the results of Example 10, where C1 is the ADORA2A gene, C2 is the CREB5 gene, C3 is the IFNGR1 gene, and C4 is the NFKB1 gene. According to the data in 3 For example, the relative expression fold value in Example 10 is close to 1, and the gene expression levels of the four genes show slight variations. Therefore, Example 10 shows the best result of preserving nucleic acids in whole blood among these examples.

Isolate nucleic acids

Example 12

cleaning of whole RNA from whole blood

Three Various methods have been used to isolate total RNA used fresh whole blood. First, RNA was prepared using Red Blood Cell Lysis Buffer (Roche Diagnostics GmbH) with a phenol-containing Reagent (Trizol Reagent, Life Technologies) according to the instructions in the manual isolated method recommended to the supplier. Secondly was the PAXgene Blood RNA Validation Kit (QIAGEN GmbH) according to the manual used by the supplier. Finally, the cleaning of the entire RNA with a surface-active primary Amine compound as described below.

First was a lysis buffer consisting of 1% (w / v) dodecylamine, 50mM maleic and 3% (v / v) Tween 20. Fresh human thoroughbred (333 μl) was mixed with 1 ml of the prepared lysis buffer and the mixture then at a rate of 11 rpm for a period of 20 minutes to Guarantee of homogeneity turned. The mixture was centrifuged at 5000 xg for 10 minutes and the supernatant then rejected; the pellet was filled with 333 μl of double distilled water washed. Then the solution containing the pellet was washed with 5000 xg for centrifuged for a period of 10 minutes and the supernatant completely discarded.

147 μl buffer RLT (QIAGEN GmbH) was added and mixed thoroughly with the sample. Then 200 μl Added 1-bromo-3-chloropropane and for a period of 10 seconds whirled. The sample was run at 10,000 xg for a duration of 5 minutes centrifuged and the supernatant then transferred to a new tube. 90 μl 100% Ethanol was added and mixed by pipetting three times and the solution then in a RNeasy mini column (QIAGEN GmbH).

The Sample was for 13000 rpm for centrifuged for 30 seconds, the flow then discarded; 350 μl buffer RW1 became the pillar added and the mixture again at 13000 rpm for a further period of 30 Centrifuged for a few seconds. The flow was then discarded. 10 μl DNase I stock solution (QIAGEN GmbH) was added to 70 μl Buffer RDD added. The DNase I incubation mixture was added directly to the RNeasy mini column added and kept at 20-30 ° C for a duration incubated for 15 minutes; 350 μl Buffers RW1 became the column added, then with 13000 rpm for Centrifuge for 30 seconds and discard the flow.

500 μl buffer RPE (QIAGEN GmbH) were the column then added the sample at 13000 rpm for 30 seconds centrifuged and the flow discarded. Another 500 μl buffer RPE became the RNeasy column added. The pillar was rated 13000 rpm for centrifuged for a period of 30 seconds and then the flow discarded. The empty pillar was rated 13000 rpm for centrifuge for 1 minute and then place the column in a clean tube transferred to a volume of 1.5 ml. 40 μl RNase-free Water became the pillar added and the column with 13000 rpm for centrifuged for 1 minute. Another aliquot of 40 μl RNase-free Water was added and the column with 13000 rpm for centrifuged for a duration of 1 minute. 80 μl eluted solution was added to a new tube transferred to a volume of 1.5 ml.

Example 13

Purification of genomic DNA in whole blood

Three different methods were used to isolate genomic DNA from fresh whole blood for comparison. In the first method, DNA was isolated using the Red Blood Cell Lysis Buffer (Roche Diagnostics GmbH) and a phenol-containing reagent (Trizol Reagent, Life Technologies) following the procedures recommended in the manual as recommended by the supplier. The second method used the QIAamp DNA Blood Kit (QIAGEN GmbH) according to the supplier's manual. Finally, in the third method, the purification of the entire DNA using a surface-active pri amine compound as described below.

333 μl fresh Whole blood were mixed with 1 ml of a solution consisting of 1% (w / v) dodecylamine, 3% (v / v) Tween 20 and 50 mM maleic acid, mixed. To isolate the DNA, the complexes of the surface active primary Amine compound and the DNA at 5000 xg for a period of 10 minutes centrifuged. The pellet was placed in a mixture of 200 μl buffer AL (QIAGEN GmbH) and 20 μl Proteinase K solved. After incubation at 55 ° C for one For 10 minutes, the sample was mixed with 200 μl of ethanol and then applied to a spin column containing a silica membrane. The sample mixture was passed through the membrane under centrifugation. The silica membrane was used to elute the DNA molecules with 500 μl Buffer AW1 (QIAGEN GmbH), then with 500 μl buffer AW2 (QIAGEN GmbH) and after all with 200 μl washed with distilled water.

Example 14

Quality and quantities of nucleic acids

To the Evaluating the concentrations of isolated nucleic acids were Measurements were made with a spectrophotometer in OD260 / 280. In Referring to Table 3, there is a comparison table of quality and quantities represented by three different isolation methods. The three Methods are: A. isolation with a primary amine of the present invention, B. commercially available Kits of PAXgene Blood RNA Validation Kit or QIAamp DNA Blood Kit and C. Isolation with conventional Method using RBC lysis and Trizol reagent.

Table 3

On the basis of the data of Table 3 showed the isolation of RNA with a primary Amine of the present invention, the highest amount of these processes, and the quality was also in an ideal range of 1.9 to 2.1. The data showed that the surface active primary Amine compound of the present invention also in DNA isolation useful was. The obtained samples were further used checked by agarose gel electrophoresis.

As in 4 Lane D1 represented the DNA isolation result by a conventional method of the RBC lysis and trizol reagents; Lane D2 demonstrated the DNA isolation result by QIAamp DNA Blood Kit; and Lane D3 represented the DNA isolation result by a surface-active primary amine of the present invention. Lane R1 represented the RNA isolation result by the conventional method of the RBC lysis and trizol reagent; Lane R2 represented the RNA isolation result by PAXgene RNA Blood Kit; and Lane R3 represented the RNA isolation result by the primary amine surface active compound of the present invention. The amount of each loading lane was 100 ng of DNA or 200 ng of RNA. The results of Lane D3 and Lane R3 indicated that the quality of the isolated nucleic acids by the primary amine surface active compound was more efficient than in the other two conventional methods.

The Results showed that the use of a surfactant primary Amine compound DNA molecules with good quality in a good quality way can isolate. The amount of isolated RNA in lane R3 was about same as in the other two tracks. It is clear from course R3 shown that with the method of isolating RNA with a surface active primary Amine compound pure RNA molecules can be obtained with genomic DNA contaminants.

Example 15

Cleaning of whole RNA with a surface active secondary Amine compound.

18 solutions 1%, 2% or 3% N-methyldodecylamine surfactant and tartaric acid in various concentrations of 25, 50, 75, 100, 125 or 150 mM were prepared. 1 ml of each surfactant was separated 333 μl fresh Added blood, the sample mixtures were at a rate from 11 rpm for rotated for 20 minutes at room temperature.

The solution was for centrifuged for 10 minutes at 5000 xg. The supernatant was then removed by decanting or pipetting. 666 μl double Distilled water was then added to the pellet and the pellet again by thorough Suspended whirling. The sample was for a duration of 10 minutes centrifuged at 5000 xg. The entire supernatant was then removed and discarded.

The Pellet was thoroughly cleaned again with 50 μl RNase-free water by pipetting until no more particles were seen, suspended. Then, 100 μl buffer RLT (Qiagen, RNeasy Minituit) and 40 μl Proteinase K added and mixed evenly by pipetting. The solution was for a duration of 10 minutes at 55 ° C in a shaking incubator or water bath. 200 μl 1-bromo-3-chloropropane were added to the sample and mixed by vortexing. The sample was then for centrifuged for 5 minutes at 10,000 xg. The supernatant was in a new tube transferred with a volume of 1.5 ml.

90 μl 100% Ethanol was added to the sample and mixed by vortexing. The mixture was then reduced to remove droplets in the tube lid Speed centrifuged briefly (less than 2 seconds).

The sample solutions were placed on a RNeasy mini column upset and for centrifuged for 30 seconds at 13000 rpm. Then were 350 μl buffer RW 1 of the column added and for centrifuged for 1 minute at 13000 rpm. The old one Flow-containing processing tubes were discarded.

10 μl of DNase I stock solution added and with 70 .mu.l Buffer RDD mixed. The resulting 80 ul DNase I incubation mixture were then transferred directly to the spin column membrane, and the sample was for allowed to stand for 15 minutes at room temperature. 350 μl buffer RW 1 was applied to the column, and the spin column was for centrifuged for 30 seconds at 13000 rpm. The old one Flow-containing processing tubes were discarded. 500 μl buffer RPE were applied to the column, and the pillar was for centrifuged for 30 seconds at 13000 rpm. The old the flow-containing processing tube was discarded.

Further 500 μl buffer RPE were in the column added. The pillar was then for centrifuged for 1 minute at 13000 rpm. The old one Flow-containing processing tubes were discarded. The Pillar became again for centrifuged for 2 minutes at 13000 rpm.

The Pillar became in a new elution tube transferred with a volume of 1.5 ml, after which 40 μl RNase-free Water directly on the column membrane were pipetted and the sample tube for a period of 1 minute with Centrifuged at 13000 rpm. Another 40 ml of RNase-free water were applied to the column centrifuged again, and the RNA product was left after the Centrifugation in an elution tube flow.

The Pellet size (%) achieved by the methods illustrated above isolated RNA products is shown in Table 4A.

Table 4A

The Pellet size was as the ratio of the volume of the pellet to the volume of blood. The higher value the pellet size indicates indicating that more contaminants contained in the pellet or Contaminants templates.

table 4B shows the RNA yield (μg / ml Blood) after RNA isolation according to the above procedure.

Table 4B

On removed the base of the data shown in Tables 4A and 4B the formulation of 2% or 3% N-methyldodecylamine with 75 to 150 mM tartaric acid the largest amount contaminants or contaminants in the isolated RNA product, and the yields were satisfactory.

Example 16

Cleaning of whole RNA with surface-active tertiary Amine.

10 solutions of 1% or 3% N, N-dimethyldodecylamine with tartaric acid in various concentrations of 50, 75, 100, 125 or 150 mM were prepared. 333 μl fresh Blood was then mixed with 1 ml of each of the surfactants prepared solutions mixed, and the obtained sample mixture was at room temperature at a rate of 11 rpm for a duration of 20 minutes to ensure homogeneity turned. The solution was then for centrifuged for 10 minutes at 5000 xg. The supernatant was removed by decanting or pipetting. Then 666 μl were duplicated distilled water was added to the pellet, and the pellet was added again thoroughly suspended by stirring. The mixture was for a duration centrifuged for 10 minutes at 5000 xg. The entire supernatant was then removed and discarded.

The Pellet was thoroughly with 167 μl Buffer RLT (Qiagen, RNeasy minikit) until no particle is visible was resuspended. 200 μl 1-bromo-3-chloropropane were added to the sample and mixed by vortexing. The sample was then for centrifuged for 5 minutes at 10,000 xg and the supernatant in a new tube transferred with a volume of 1.5 ml.

90 μl 100% Ethanol was added to the sample and mixed by vortexing. The mixture was then used to remove droplets in the tube cap at low speed centrifuged briefly (less than 2 seconds).

The sample solutions were placed on a RNeasy mini column upset and for centrifuged for 30 hours at 13000 rpm. Then were 350 μl buffer RW1 of the column added and for centrifuged for 1 minute at 13000 rpm. The old one Flow-containing processing tubes were discarded.

10 μl of DNase I stock solution to 70 μl Buffer RDD added. The DNase I incubation mixture (80 μl) was added then transferred directly into the spin column membrane and the sample at room temperature for a period of 15 minutes left. 350 μl buffer RW1 was added to the Pillar applied and for centrifuged for 30 seconds at 13000 rpm. The old the flow-containing processing tube was discarded. 500 μl buffer RPE were on the pillar applied, and the column was for centrifuged for 30 seconds at 13000 rpm. The old the flow-containing processing tube was then discarded.

Further 500 μl buffer RPE became the pillar added. The pillar was then for centrifuged for 1 minute at 13000 rpm and the old one the flow-containing processing tube then discarded. The Pillar became again for centrifuged for 2 minutes at 13000 rpm.

The Pillar became then into a new elution tube transferred with a volume of 1.5 ml, after which 40 ul RNase-free Water directly on the column membrane were applied and the column tube for a duration centrifuged for 1 minute at 13000 rpm. Another 40 μl RNase-free Water was on the column applied and again centrifuged.

The Pellet size (%) and the yields of the isolated RNA products by the ones illustrated above Methods are shown in Tables 5A and 5B, respectively.

Table 5A

Table 5B

On removed the base of the data shown in Tables 5A and 5B the formulation of 3% N, N-dimethyldodecylamine with 75 mM tartaric acid effi cient the contaminants or contaminants in the isolated RNA, what to the highest quality which resulted in RNA products.

The isolation efficiency is shown in the electrophoresis results in 5 in which lane 1 to lane 5 were the lanes in which 1% N, N-dimethyldodecylamine was used and lane 6 to lane 10 were the lanes in which 3% N, N-dimethyldodecylamine was used. Lanes 1 and 6, 2 and 7, 3 and 8, 4 and 9, 5 and 10 were formulated separately with 50 mM, 75 mM, 100 mM, 125 mM and 150 mM tartaric acid.

Example 17

Cleaning of whole RNA with surface-active tertiary Amine oxide.

16 Solutions, in which each 1% N, N-dimethyldodecylamine N-oxide with different Amounts of maleic acid, Citric acid, tartaric acid or oxalic acid were prepared in concentrations of 25, 50, 75 or 100 mM. Then the steps described above for isolation followed by RNA from a whole blood sample in Example 16.

table 6 shows the RNA yields (μg / ml Blood) after RNA isolation by following the above procedures.

Table 6

The isolation efficiency is from the eletrotrophoresis result in 6 where Part A is the result of maleic acid, Part B is the result of citric acid; in Part C, the result was treated with tartaric acid and in Part D the result was oxalic acid. Lane 1 was at a concentration of 25mM, Lane 2 at 50mM, Lane 3 at 75mM and Lane 4 at 100mM.

According to the data in Table 6 and the result of eletrophoresis in 6 All of the RNA samples isolated with 1% N, N-dimethyldodecylamine N-oxide and citric acid, tartaric acid or oxalic acid showed good quality and acceptable levels.

Example 18

Purification of genomic DNA in whole blood with 4-tetradecylaniline.

3 solutions of 1% 4-tetradecylaniline and Tween 20 at various concentrations 3, 4 or 5% were prepared. 333 μl of fresh whole blood were mixed with 1 ml of each of the surfactant solutions prepared. The mixture was then taken at 5000 xg for a period of 10 minutes centrifuged to the complexes of DNA molecules and the surface active primary Precipitate amines. The pellet was dissolved in 333 μl Buffer RLT (QIAGEN GmbH) solved. Then 200 μl 1-bromo-3-chloropropane added to the sample and mixed by vortexing. The sample was then used for centrifuged for 5 minutes at 10,000 xg.

The supernatant was in a new tube transferred with a volume of 1.5 ml, then with 160 μl of ethanol mixed and applied to a spin column containing a silica membrane. The sample was passed through the membrane under centrifugation. The silica membrane was used to elute the DNA molecules once 700 μl buffer RW1 (QIAGEN GmbH), then twice with 500 .mu.l buffer RPE (QIAGEN GmbH) and finally with 80 μl distilled Washed water.

The isolation efficiency is from the electrophoresis result in 7 The three lanes are shown with different concentrations of Tween 20. Lane 1 was at 3% concentration, Lane 2 at 4% concentration and Lane 3 at 5% concentration. According to the results, the DNA samples isolated with 4-tetradecylaniline showed good quality and satisfactory amount. In addition, intact genomic DNA can be extracted.

The embodiments show that nucleic acids in a biological sample with different stabilizing solutions, containing primary Amine, secondary Amine or tertiary Amine of the present invention, effectively stabilized or isolated can be. The present invention extends the stabilizing or Conservation periods and also improves the amount of insulation with simple procedures according to the data the examples described above. The procedure can be automatic carried out to increase throughput and the application of molecular diagnostic testing with nucleic acids expand.

Even though the present invention with respect to its preferred embodiment has been described, it is clear that many other possible modifications and variations without leaving the as claimed below Spirit and scope of the invention can be performed.

Claims (28)

A method of stabilizing or isolating nucleic acids by forming an insoluble ionic one A complex between nucleic acids and a surface-active agent in a biological sample, comprising a step of contacting the biological sample with a stabilizing or insulating composition comprising amine-surface active compounds of formula (I): R ₁ R ₂ R ₃ N (O) _x , (I) wherein R ₁ and R _{2 are} each independently H, a C 1 -C 6 alkyl group, C 6 -C 12 aryl group or C 6 -C 12 aralkyl group; R _{3 is} a C 1 -C 20 alkyl group, C 6 -C 26 aryl group or C 6 -C 26 aralkyl group; and x is an integer of 0 or 1. The process of claim 1 wherein x is 1, R ₁ and R _{2 are} each independently C ₁ -C 6 alkyl and R _{3 is} C ₁ -C 20 alkyl. The method of claim 1, wherein x is 0 The method of claim 1, wherein the surfactant Amine compounds selected are from the group consisting of dodecylamine, N-methyldodecylamine, N, N-dimethyldodecylamine, N, N-dimethyldodecylamine N-oxide and 4-tetradecylaniline. The method of claim 1, wherein the concentration the surface active Amine compounds in the composition in the range of 0.001 to 20% lies. The method of claim 1, wherein the composition further at least one nonionic detergent, at least an acid salt or the mixture thereof. The method of claim 6, wherein the at least one nonionic detergent is polyoxyethylene sorbitan monolaurate. The method of claim 6, wherein the at least one nonionic detergent in the composition in the range of 0.01 up to 20%. The method of claim 6, wherein the at least one non-ionic detergent Tween 20 or Triton X-100. The method of claim 6, wherein the at least an acid salt selected is from the group consisting of maleic acid, tartaric acid, citric acid, oxalic acid, carboxylic acids and Mineral acids. The method of claim 6, wherein the total concentration the acid salts in the composition ranges from 0.01 M to 1 M. The method of claim 1, wherein the pH of the Composition is in the range of 1-10. The method of claim 1, wherein the biological Sample selected is from the group consisting of whole blood, plasma, serum, urine, Tissues and cells. The method of claim 1, wherein the nucleic acids are DNA and RNA are. A reagent for stabilizing or isolating nucleic acids in a biological sample, comprising amine-surface active compounds of the formula (I): R ₁ R ₂ R ₃ N (O) _x , (I) wherein R ₁ and R _{2 are} each independently H, a C 1 -C 6 alkyl group, C 6 -C 12 aryl group or C 6 -C 12 aralkyl group; R _{3 is} a C 1 -C 20 alkyl group, C 6 -C 26 aryl group or C 6 -C 26 aralkyl group; and x is an integer of 0 or 1. A reagent according to claim 15 wherein x is 1, R ₁ and R _{2 are} each independently C ₁ -C 6 alkyl and R _{3 is} C ₁ -C 20 alkyl. A reagent according to claim 15, wherein x is 0 A reagent according to claim 15, wherein the surfactant Amine compounds selected are from the group consisting of dodecylamine, N-methyldodecylamine, N, N-dimethyldodecylamine, N, N-dimethyldodecylamine N-oxide and 4-tetradecylaniline. A reagent according to claim 15, wherein the concentration the surface active Amine compounds in the reagent is in the range of 0.001 to 20%. A reagent according to claim 15, further at least a nonionic detergent, at least one acid salt or the mixture thereof full. A reagent according to claim 20, wherein the nonionic Detergent is polyoxyethylene sorbitol monolaurate. A reagent according to claim 20, wherein the nonionic Detergents in the reagent in the range of 0.01 to 20%. A reagent according to claim 20, wherein the nonionic Detergent Tween 20 or Triton X-100. A reagent according to claim 20, wherein said at least one acid salt selected is from the group consisting of maleic acid, tartaric acid, citric acid, oxalic acid, carboxylic acids and Mineral acids. A reagent according to claim 20, wherein the total concentration the acid salts in the reagent ranges from 0.01M to 1M. A reagent according to claim 15, wherein the pH of the Reagenses in the range of 1-10. A reagent according to claim 15, wherein the biological Sample selected is from the group consisting of whole blood, plasma, serum, urine, Tissues and cells. A reagent according to claim 15, wherein the nucleic acids are DNA and RNA are.