EP1268769A2

EP1268769A2 - Method for extracting nucleic acids

Info

Publication number: EP1268769A2
Application number: EP01936163A
Authority: EP
Inventors: Andreas Bosio
Original assignee: Memorec Stoffel Medizinisch-Molekulare Entwicklung GmbH
Current assignee: Memorec Biotec GmbH
Priority date: 2000-03-31
Filing date: 2001-03-30
Publication date: 2003-01-02
Also published as: WO2001072998A3; AU2001262152A1; WO2001072998A2; US20040142324A1; US20080009614A1; JP2003530094A

Abstract

A nuclear acid complex comprising an anchor nucleotide, any particular nucleic acid or any particular oligonucleotide and a linker nucleic acid. The linker is partially hybridized with the anchor nucleotide and partially hybridized with the any particular nucleic acid or oligonucleotide. The anchor nucleotide is immobilized at the 3,-terminus on a support.

Description

Process for the extraction of nucleic acids

The present invention relates to a nucleic acid complex with an anchor oligonucleotide, any nucleic acid or any oligonucleotide and a linker oligonucleotide, a nucleic acid or oligonucleotide hybridization product and a method for producing and separating nucleic acids or oligonucleotides after their synthesis using the nucleic acid complex according to the invention.

The invention further relates to a device which makes it possible to extract, purify, rewrite, rewrite and re-purify RNA from homogenized tissue or cells in a combined and automatable one-tube reaction.

The analysis of gene expression requires the extraction, purification and labeling of the mRNA. The labeling of the mRNA is often carried out via the step of cDNA synthesis, since labeled nucleotides can be inserted here. The process of mRNA isolation includes the selective extraction of the RNA from the mixture of the materials present in a cell or in tissue (lipids, proteins, saccharides, DNA, RNA, low-molecular substances) and the subsequent enrichment of the mRNA from the mixture of tRNA, rRNA, snRNA and mRNA with the help of oligo (dT) nucleic acids, which are bound to a column (affinity chromatography) or to (magnetic) spherical particles (batch process). The mRNA is then eluted from the oligo (dT) and subjected to further enzymatic reactions separately therefrom, or is still enzymatically converted to oligo (dT) (and a solid phase). The RT reaction for the production of the cDNAs is an enzymatic reaction which can also be carried out in the presence of the oligo (dT) and a solid phase coupled to it. However, the synthesized cDNA cannot easily be separated from the solid phase, since the oligo (dT) s serve as primers and the cDNA is thus ultimately covalently bound to the solid phase. Another It is therefore not possible to use the cDNA, which requires this to be separated from the solid phase.

WO-A-98/31838 relates to a method for identifying gene expression patterns in mRNA populations. The method is useful for determining differential gene expressions in different cells or tissues, including cells or tissues of target organisms. Methods are disclosed for determining the frequency of gene expression in mRNA populations, whereby a method for comparing the frequency of gene expression of different cells or tissues becomes available. Also described is a method for isolating genes that were identified using so-called tag sequences according to the method described there. The sequences can be used to diagnose certain diseases.

WO-A-95/13368 relates to the isolation of a nucleic acid from a sample. The sample is boiled and then cooled. The nucleic acids are condensed on a solid support. The method is used to prepare nucleic acids for subsequent amplification. In particular, the method can be used in the isolation of nucleic acids from aged, fixed or otherwise treated samples.

WO-A-97/10363 relates to the Serial Analysis of Gene Expression (SAGE), a method for the rapid, quantitative and qualitative analysis of transcripts. Short, defined sequences that correspond to expressed genes are isolated and analyzed. The sequencing of over 1000 defined tags in a short time results in a gene expression pattern that is characteristic of the function of a cell or a tissue. The SAGE method is useful as a tool for identifying and isolating new tag sequences that correspond to new transcripts and genes.

The technical problem on which the invention is based is to provide a simple and reliable method and means for carrying out the method in order to avoid the disadvantages described above. The object is achieved by a nucleic acid complex with an anchor oligonucleotide, any nucleic acid or any oligonucleotide as well as a linker oligonucleotide, the linker oligonucleotide being partially hybridized with the anchor oligonucleotide and partially with the arbitrary nucleic acid or any oligonucleotide and the anchor oligonucleotide with its 3 ^λ -T is immobilized on a carrier.

The anchor oligonucleotide is immobilized on a support. The linker oligonucleotide has a sequence which allows hybridization both with the anchor oligonucleotide and with a further nucleic acid or a further oligonucleotide. The sequence of the linker oligonucleotide must therefore be chosen so that it matches the sequence of the further nucleic acid / the further oligonucleotide. If, for example, cDNA is to be obtained from mRNA (poly (A ⁺ ) RNA), the linker oligonucleotide has a poly (dT) part.

The method according to the invention for producing cDNA is explained in more detail with reference to FIG. 2:

A carrier, preferably a bead, is used, to which a linker oligonucleotide is bound via an anchor oligonucleotide in such a way that a poly (dT) part is present. MRNA is now attached to this poly (dT) part, and the linker oligonucleotide is lengthened by a polymerase reaction by means of a conventional RT reaction - if appropriate with the incorporation of labeled nucleotides. The mRNA can then be destroyed, for example by RNaseH or NaOH, and the linker oligonucleotide extended by the cDNA can be cleaved off by increasing the temperature, if necessary by changing the buffer.

The method can also be used to synthesize any nucleic acid (with an at least partially known sequence). This is it required that the linker oligonucleotide again has a sequence that partially overlaps with the target molecule. Then a nucleic acid complementary to the target nucleic acid can be synthesized on the linker oligonucleotide, possibly with the incorporation of labeled nucleotides. The nucleic acid can then be split off as a double strand (with a single strand end) and processed further. Such a method is useful, for example, for the direct, non-PCR-amplified production of cDNA libraries with selectively enriched cDNA molecules in accordance with the selected sequence of the linker oligonucleotide, by ligating the isolated and double-stranded cDNAs directly into a suitably prepared cloning vector ,

In the nucleic acid complex according to the invention, z. B. a temperature of 35 ° C to 85 ° C, preferably 45 ° C to 65 ° C, for annealing of anchor oligonucleotide and linker oligonucleotide.

The hybridizing region of anchor and linker oligonucleotide in the nucleic acid complex according to the invention preferably has a ratio of the nucleotides GC: AT of 20:80 to 80:20.

According to the invention, annealing between anchor oligonucleotide and linker oligonucleotide is preferably carried out at a higher temperature than between linker oligonucleotide and any nucleic acid or oligonucleotide.

In a preferred embodiment, the nucleic acid complex according to the invention has the anchor oligonucleotide covalently bound or immobilized on the support via an affinity group.

The present invention also relates to a nucleic acid or oligonucleotide hybridization product comprising an anchor oligonucleotide and a linker oligonucleotide, the linker oligonucleotide being partially hybridized with the anchor oligonucleotide and the anchor oligonucleotide with the 3'- Term can be immobilized on a carrier.

The anchor oligonucleotide and the linker oligonucleotide, which can be immobilized on the support with the 3'-terminus, can be sold in the form of a kit, optionally together with other components for carrying out the method according to the invention.

The invention also relates to a method for producing and separating nucleic acids or oligonucleotides after their synthesis using an embodiment of the nucleic acid complex according to the invention. The following steps are carried out:

Addition of any target nucleic acid whose 5 'terminus partially hybridizes with the linker oligonucleotide, the linker oligonucleotide being hybridized with the anchor oligonucleotide immobilized on a support,

• Extension of the linker oligonucleotide by a polymerase and

• Detachment of the extended linker oligonucleotide together with the target nucleic acid if necessary.

The target nucleic acid is preferably mRNA. The target nucleic acid can be degraded by RnaseH or NaOH, in particular before the extended linker oligonucleotide is split off.

With the preferred linker and anchor nucleotide shown in FIG. 1, the invention allows a temporary non-covalent binding of the cDNA to the solid phase, which can be split off by a simple heat step. This means that all reactions required for the isolation, purification, marking and renewed cleaning of the finished probe can be carried out on the solid phase (FIG. 2). The implementation on the solid phase in turn enables simple automation of the complete process (FIG. 3). The invention is illustrated by the following examples. The method according to the invention was used to produce fluorescence-labeled cDNA starting from total RNA. Dynabeads M-280 streptavidin (DYNAL) was used as the solid phase. Different quality controls were carried out to check the mRNA yield, incorporation efficiency and purity of the Cy5-labeled cDNA. The labeled cDNA was then hybridized with a cDNA (Cy3) labeled in a conventional manner with a cDNA array. The conventional labeling was carried out as follows: incorporation of fluorescence-labeled Cy3 nucleotides in solution during a reverse transcriptase reaction (Superscript II (GIBCO) starting from purified mRNA which was isolated from the same total RNA as was used for the other probe, Subsequent cleavage of the mRNA by RnaseH treatment and purification of the labeled cDNA over silica membranes (Qiaquick, QIAGEN) On the basis of the Cy3 / Cy5 signal ratio of the individual cDNAs immobilized on the array, it was possible to make a statement about the quality of the labeling method according to the invention compared to the conventional one Marking procedures are carried out as follows:

a) Production of the anchor-linker hybrid:

The biotin anchor and linker were adjusted to a concentration of 350 ng / μl in RNase-free, double-distilled water. In a 0.2 ml Eppendorf reaction vessel, 9.1 μl biotin anchor (400 pmol; 8000 pg / pmol), 15.6 μl linker (400 pmol; 13647 pg / pmol) and 25 μl of a solution of 10 mM Tris-HCl, pH 7.5; 1 mM EDTA and 2 M NaCI combined and incubated for 2 min at 95 ° C, 10 min at 65 ° C, 10 min at 37 ° C and 20 min at RT. To check that the hybrid formation had taken place, 1 μl of the solution was removed and 4 μl 6 × PAGE loading buffer and 19 μl H ₂ O were combined, separated using a 12% PAGE gel (19: 1 acrylamide: bisacrylamide) and with a 1: 1000 diluted SybrGreen (Molecular Probes) stained. In parallel 1 μl of the biotin anchor and 1 μl of the linker were applied, see FIG. 4. While the single-strand linker runs at a height of 25 bp and the single-strand biotin anchor runs at a height of 35 bp (the biotinylation of this single strand does not necessarily result in one clean separation), it can be clearly seen that the anchor-linker hybrid runs at a height of almost 50 bp.

b) Pretreatment of Dynabeads M-280 straptavidin (according to the manufacturer):

200 μl (2 mg) of Dynabeads M-280 straptavidin were washed once with twice the volume of a solution of 0.1 M NaOH and 0.05 M NaCI, once with twice the volume of a 0.1M NaCI solution and in 375 μl of a solution of 10 mM Tris-HCl , pH 7.5; 1 mM EDTA and 2 M NaCI resuspended.

c) Immobilization of the anchor-linker hybrid:

375 μl of a solution of 3 mM Tris-HCl (pH7.5) are added to the solution obtained under b)

0.2 mM EDTA and 50 μl of the anchor-linker hybrids obtained under a) were added and incubated for 15 min at RT with occasional shaking. Using the magnetic stand provided for this purpose (DYNAL), twice with a solution of 10 mM Tris-HCl, pH 7.5; 1 mM EDTA and 2 M NaCl and then resuspended in 200 μl of a solution of 20 mM Tris-HCl, pH 7.5, 1 M LiCI, 2 mM EDTA.

d) mRNA isolation:

100 μg of total RNA were heated to 65 ° C. in 100 μl of H ₂ O for 2 min and then added to the derivatized beads obtained from c) and freed from the solution. The reaction vessel was at RT for 5 min shaken, placed in the magnetic stand for 5 min and then the beads washed twice with 200 μl of a solution of 10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA.

e) Labeling reaction:

The isolated mRNA was washed once with a solution of 3 mM Tris-HCl (pH7.5), 0.2 mM EDTA and 2 with 1 x RT buffer (GIBCO). 21 μl H ₂ O, 8 μl 5 × First Strand Buffer (GIBCO), low C dNTPs (10 mM dATP, 10 mM dGTP, 10 mM dTTP; 4 mM dCTP) (GIBCO), 2 μl FluoroLink ™ were placed in an Eppendorf reaction vessel Cy3 / 5-dCTP (Amersham Pharmacia), 4 μl 0.1 M DTT and 1 μl Rnasin (20-40 u) (PROMEGA) combined and shaken briefly. The isolated mRNA was resuspended with this solution, incubated for 5 min at 42 ° C., provided with 1 μl (200 U) Superscript II (SSII, GIBCO), incubated for 30 min with constant shaking at 37 ° C., again with 1 μl SSII and shaken again at 37 ° C for 30 min. 0.5 ul RNAseH (GIBCO) was added and incubated at 37 ° C for 20 min.

f) Cleaning the marked probe:

If both the Cy3 and the Cy5-labeled probe are to be produced in the manner according to the invention, then both labeling reactions could now be given together and processed further together. In the specific example described here, in which the Cy5-labeled probe was produced by the method according to the invention and, for comparison, the Cy3-labeled probe by the conventional method, the two labeling approaches were worked up separately from one another. The probe produced by the method according to the invention was washed twice with 400 μl 10 mM Tris-HCl pH 7.5, resuspended in 10 μl 10 mM Tris-HCl pH 7.5, incubated for 2 min at 65 ° C. and immediately placed in the magenta stand. The supernatant was transferred to a new Eppendorf reaction vessel. The beads were resuspended in 10 μl of 10 mM Tris-HCl pH 7.5, incubated for 2 min at 65 ° C. and the The supernatant was again transferred to the new Eppendorf reaction vessel. The combined supernatants were combined with the Cy3 probe labeled in a conventional manner, evaporated to 20 μl, provided with 5 μl of a hybridization solution and applied to a pre-hybridized cDNA array. The cDNA array was hybridized overnight and then washed with suitable solutions, dried and read out with a laser scanning device. The images (Cy3 and Cy5) obtained at different wavelengths are shown in FIG. 5. The evaluation of the relative signal intensities showed that, starting from the same amount of total RNA, the probe labeled by the method according to the invention gave an average 2.2 times stronger signals than the conventionally labeled probe, FIG. 6. The evaluation further showed that after Comparison of the absolute signal intensities with a constant normalization factor, which was calculated from the median of the signal quotients of all signal quotients, none of the array elements hybridized on the cDNA array gave a signal quotient of> ± 2, FIG. 7. Thus, the variance is not greater than at the comparison of two probes that were labeled differently from the same RNA using the conventional method using different fluorophores and hybridized on the same array.

Claims

claims

1. Nucleic acid complex with an anchor oligonucleotide, any nucleic acid or any oligonucleotide as well as a linker oligonucleotide, wherein the linker oligonucleotide is partially hybridized with the anchor oligonucleotide and partially with any nucleic acid or any oligonucleotide and the anchor oligonucleotide with its 3 ^λ terminus is immobilized on a carrier is.

2. Nucleic acid complex according to claim 1, wherein a temperature of 35 ° C to 85 ° C, preferably 45 ° C to 65 ° C, leads to an annealing of anchor oligonucleotide and linker oligonucleotide.

3. Nucleic acid complex according to one of claims 1 or 2, wherein the hybridizing region of anchor and linker oligonucleotide has a ratio of the nucleotides GC: AT of 20:80 to 80:20.

4. Nucleic acid complex according to one of claims 1 to 3, wherein an annealing between anchor oligonucleotide and linker oligonucleotide takes place at a higher temperature than between linker oligonucleotide and any nucleic acid or oligonucleotide.

5. Nucleic acid complex according to one of claims 1 to 4, wherein the anchor oligonucleotide is immobilized covalently or via an affinity group on the support.

6. Nucleic acid or oligonucleotide hybridization product comprising an anchor oligonucleotide and a linker oligonucleotide, the linker oligonucleotide being partially hybridized with the anchor oligonucleotide and the anchor oligonucleotide having the 3 'terminus being immobilizable on a support.

7. A method for producing and separating nucleic acids or oligonucleotides after their synthesis using a nucleic acid complex according to one of claims 1 to 5 or a hybridization product according to claim 6, the following steps being carried out:

Addition of any target nucleic acid whose 5 <'terminus partially hybridizes with the linker oligonucleotide, the linker oligonucleotide being hybridized with the anchor oligonucleotide immobilized on a support,

• Extension of the linker oligonucleotide by a polymerase and

• Detachment of the extended linker oligonucleotide, possibly together with the target nucleic acid.

8. The method of claim 7, wherein the target nucleic acid is mRNA.

9. The method of claim 8, wherein the target nucleic acid is degraded by RnaseH or NaOH prior to cleavage of the extended linker oligonucleotide.

10. Kit for performing the method according to one of claims 7 to 9 with

- At least one anchor oligonucleotide that is immobilized with its 3'-terminus on a support and

- At least one linker oligonucleotide.