EP1268769A2 - Procede d'extraction d'acides nucleiques - Google Patents

Procede d'extraction d'acides nucleiques

Info

Publication number
EP1268769A2
EP1268769A2 EP01936163A EP01936163A EP1268769A2 EP 1268769 A2 EP1268769 A2 EP 1268769A2 EP 01936163 A EP01936163 A EP 01936163A EP 01936163 A EP01936163 A EP 01936163A EP 1268769 A2 EP1268769 A2 EP 1268769A2
Authority
EP
European Patent Office
Prior art keywords
oligonucleotide
nucleic acid
linker
anchor
linker oligonucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01936163A
Other languages
German (de)
English (en)
Inventor
Andreas Bosio
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Memorec Biotec GmbH
Original Assignee
Memorec Stoffel Medizinisch-Molekulare Entwicklung GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Memorec Stoffel Medizinisch-Molekulare Entwicklung GmbH filed Critical Memorec Stoffel Medizinisch-Molekulare Entwicklung GmbH
Priority to EP01936163A priority Critical patent/EP1268769A2/fr
Publication of EP1268769A2 publication Critical patent/EP1268769A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Definitions

  • 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).
  • 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.
  • 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.
  • 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.
  • SAGE Serial Analysis of Gene Expression
  • 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 ⁇ -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.
  • a carrier preferably a bead
  • 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 ,
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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:
  • biotin anchor and linker were adjusted to a concentration of 350 ng / ⁇ l in RNase-free, double-distilled water.
  • a concentration of 350 ng / ⁇ l in RNase-free, double-distilled water In a 0.2 ml Eppendorf reaction vessel, 9.1 ⁇ l biotin anchor (400 pmol; 8000 pg / pmol), 15.6 ⁇ l linker (400 pmol; 13647 pg / pmol) and 25 ⁇ l 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.
  • 0.2 mM EDTA and 50 ⁇ l 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 ⁇ l of a solution of 20 mM Tris-HCl, pH 7.5, 1 M LiCI, 2 mM EDTA.
  • RNA 100 ⁇ g of total RNA were heated to 65 ° C. in 100 ⁇ l 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 ⁇ l of a solution of 10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA.
  • 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 ⁇ l H 2 O, 8 ⁇ l 5 ⁇ First Strand Buffer (GIBCO), low C dNTPs (10 mM dATP, 10 mM dGTP, 10 mM dTTP; 4 mM dCTP) (GIBCO), 2 ⁇ l FluoroLink TM were placed in an Eppendorf reaction vessel Cy3 / 5-dCTP (Amersham Pharmacia), 4 ⁇ l 0.1 M DTT and 1 ⁇ l 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 ⁇ l (200 U) Superscript II (SSII, GIBCO), incubated for 30 min with constant shaking at 37 ° C., again with 1 ⁇ l 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.
  • 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.
  • 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 ⁇ l 10 mM Tris-HCl pH 7.5, resuspended in 10 ⁇ l 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 ⁇ l 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 ⁇ l, provided with 5 ⁇ l 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Analytical Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne un complexe d'acides nucléiques comprenant un nucléotide d'ancrage, un acide nucléique quelconque ou un oligonucléotide quelconque et un acide nucléique de liaison. La séquence de liaison est partiellement hybridée avec le nucléotide d'ancrage et partiellement avec l'acide nucléique quelconque ou avec l'oligonucléotide quelconque, et le nucléotide d'ancrage est immobilisé par son extrémité 3' sur un support.
EP01936163A 2000-03-31 2001-03-30 Procede d'extraction d'acides nucleiques Withdrawn EP1268769A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP01936163A EP1268769A2 (fr) 2000-03-31 2001-03-30 Procede d'extraction d'acides nucleiques

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
DE10016138 2000-03-31
DE10016138 2000-03-31
EP00109297 2000-04-29
EP00109297 2000-04-29
PCT/EP2001/003669 WO2001072998A2 (fr) 2000-03-31 2001-03-30 Procede d'extraction d'acides nucleiques
EP01936163A EP1268769A2 (fr) 2000-03-31 2001-03-30 Procede d'extraction d'acides nucleiques

Publications (1)

Publication Number Publication Date
EP1268769A2 true EP1268769A2 (fr) 2003-01-02

Family

ID=26005130

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01936163A Withdrawn EP1268769A2 (fr) 2000-03-31 2001-03-30 Procede d'extraction d'acides nucleiques

Country Status (5)

Country Link
US (2) US20040142324A1 (fr)
EP (1) EP1268769A2 (fr)
JP (1) JP2003530094A (fr)
AU (1) AU2001262152A1 (fr)
WO (1) WO2001072998A2 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006158276A (ja) * 2004-12-06 2006-06-22 Institute Of Physical & Chemical Research Ｄｎａコンジュゲート、ｄｎａコンジュゲートの作製方法、及びｄｎａ検出方法
US20070072223A1 (en) * 2005-09-16 2007-03-29 Primera Biosystems, Inc. Compositions and methods for purifying nucleic acids
US8383338B2 (en) * 2006-04-24 2013-02-26 Roche Nimblegen, Inc. Methods and systems for uniform enrichment of genomic regions
US9340416B2 (en) * 2008-08-13 2016-05-17 California Institute Of Technology Polynucleotides and related nanoassemblies, structures, arrangements, methods and systems
KR101230913B1 (ko) * 2009-11-06 2013-02-07 중앙대학교 산학협력단 나노입자-기반된 유전자 운반체
EP3313334A1 (fr) 2015-06-29 2018-05-02 The Procter and Gamble Company Pessaire associé à un applicateur
KR101949371B1 (ko) * 2015-10-07 2019-02-18 주식회사 엘지화학 내후성 열가소성 수지, 이를 포함하는 열가소성 수지 조성물 및 이 조성물을 제조하는 방법
GB201607817D0 (en) * 2016-05-04 2016-06-15 Univ Leiden Methods

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6022714A (en) * 1985-05-02 2000-02-08 Genetics Institute Methods for attachment of a polynucleotide to a preselected material
GB9323305D0 (en) * 1993-11-11 1994-01-05 Medinnova Sf Isoaltion of nucleic acid
US5866330A (en) * 1995-09-12 1999-02-02 The Johns Hopkins University School Of Medicine Method for serial analysis of gene expression
US5837466A (en) * 1996-12-16 1998-11-17 Vysis, Inc. Devices and methods for detecting nucleic acid analytes in samples
US5968784A (en) * 1997-01-15 1999-10-19 Chugai Pharmaceutical Co., Ltd. Method for analyzing quantitative expression of genes
CA2377468C (fr) * 1999-07-27 2010-04-20 Phylos, Inc. Methodes de ligature d'un accepteur de peptide

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0172998A2 *

Also Published As

Publication number Publication date
WO2001072998A3 (fr) 2002-01-24
AU2001262152A1 (en) 2001-10-08
WO2001072998A2 (fr) 2001-10-04
JP2003530094A (ja) 2003-10-14
US20080009614A1 (en) 2008-01-10
US20040142324A1 (en) 2004-07-22

Similar Documents

Publication Publication Date Title
DE69738603T2 (de) Verfahren zur Herstellung von Banken vollständiger cDNAs
EP0743367B1 (fr) Méthode pour l'analyse de l'expression génétique
DE69825240T2 (de) Verfahren zur subtraktiven hybridisierung und differenziellen analyse
DE102008025656B4 (de) Verfahren zur quantitativen Analyse von Nukleinsäuren, Marker dafür und deren Verwendung
DE69030955T2 (de) Nukleinsäureverstärkung
DE69028725T2 (de) Partiell doppelsträngiges Oligonukleotid und Verfahren zu seiner Bildung
DE69621507T2 (de) Verfahren zur molekularen Indexierung von Genen unter Verwendung von Restriktionsenzymen
DE3687537T2 (de) Verfahren zur amplifikation, zum nachweis und/oder zur klonierung von nukleinsaeuresequenzen.
DE3855064T2 (de) Selektive Amplifikation von Oligonukleotiden-Zielsequenzen
EP1685261B1 (fr) Synthetiseur d'adn a parallelisme eleve sur une base matricielle
EP1204765B1 (fr) Procede de quantification relative de la methylation des bases du type cytosine dans des echantillons d'adn
EP1889925B1 (fr) Introduction d'éléments de séquence dans des acides nucléiques
DE69011101T2 (de) Methoden zur in vitro-dna-amplifikation, zum genomischen klonieren und zur genkartierung.
DE69219627T2 (de) Verfahren zur Bestimmung von einer Nukleinsäuresequenz
WO1989011211A2 (fr) Banque d'oligonucleotides et procede de sequençage d'adn
DE68922743T2 (de) Verfahren zur herstellung von cdns.
US20080009614A1 (en) Method for extracting nucleic acids
WO2000014275A2 (fr) Representation differentielle en deux couleurs utilise comme procede de detection de genes regules
DE3688965T2 (de) Verfahren und aufbau einer nukleinsäure zur erzeugung von reagenzienkomplexen zur verwendung bei der bestimmung von zielnukleotidsequenzen.
EP0523557A1 (fr) Immobilisation des acides nucléiques
DE602006000713T2 (de) Verfahren zur Verbesserung der Unterscheidung von einer einzelnen Basenfehlanpaarung durch die Verwendung von "hurdle" DNAs und Sonden mit artifiziellen Fehlpaarungen
DE4428651C1 (de) Verfahren zur Herstellung und Amplifikation von Nukleinsäuren
WO1994029443A1 (fr) Analyse de sequences d'arn par pcr
EP2706124B1 (fr) Mise en évidence simultanée de différentes micro-formes de biogenèse RNA
DE10314745A1 (de) Zyklische Nukleinsäure-Amplifikation mittels einer Helicase

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020925

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: MEMOREC BIOTEC GMBH

17Q First examination report despatched

Effective date: 20051021

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20080703