(19) TZZ ¥¥_T (11) EP 2 626 433 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) C12N 15/62 (2006.01) 12.04.2017 Bulletin 2017/15 C12N 15/13 (2006.01) C07K 16/00 (2006.01) (21) Application number: 12154726.9 (22) Date of filing: 09.02.2012 (54) Method for linking nucleic acids in a microsome from endoplasmatic reticuli Verfahren zum Koppeln von Nukleinsäuren in einem Mikrosom aus den endoplasmatischen Retikuli. Procédé pour lier les acides nucléiques dans un microsome provenant du réticulum endoplasmique (84) Designated Contracting States: • ANGENENDT P ET AL: "CELL-FREE PROTEIN AL AT BE BG CH CY CZ DE DK EE ES FI FR GB EXPRESSION AND FUNCTIONAL ASSAY IN GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO NANOWELL CHIP FORMAT", ANALYTICAL PL PT RO RS SE SI SK SM TR CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 76, no. 7, 1 April 2004 (2004-04-01), pages (43) Date of publication of application: 1844-1849, XP001196723, ISSN: 0003-2700, DOI: 14.08.2013 Bulletin 2013/33 10.1021/AC035114I • DATABASE MEDLINE [Online] US NATIONAL (73) Proprietors: LIBRARY OF MEDICINE (NLM), BETHESDA, MD, • Max-Planck-Gesellschaft zur Förderung US; December 2001 (2001-12), WANG M ET AL: der Wissenschaften e.V. "[Sequence analysis of the late region of human 80539 München (DE) papillomavirus type 6 genome].", XP002678283, • Alacris Theranostics GmbH Database accession no. NLM12901100 & 14195 Berlin (DE) ZHONGGUO YI XUE KE XUE YUAN XUE BAO. ACTA ACADEMIAE MEDICINAE SINICAE DEC (72) Inventors: 2001 LNKD- PUBMED:12901100, vol. 23, no. 6, • Warnatz, Hans-Jörg December 2001 (2001-12), pages 568-572, ISSN: 10245 Berlin (DE) 1000-503X • Glökler, Jörn • A. M. SMITH ET AL: "Highly-multiplexed barcode 10783 Berlin (DE) sequencing: an efficient method for parallel •Lehrach,Hans analysis of pooled samples", NUCLEIC ACIDS 14129 Berlin (DE) RESEARCH, vol. 38, no. 13, 11 May 2010 (2010-05-11), pages E142-E142, XP55017285, (74) Representative: CH Kilger Anwaltspartnerschaft ISSN: 0305-1048, DOI: 10.1093/nar/gkq368 mbB • BRAND KORBINIAN ET AL: "A novel A fwdarw G Fasanenstrasse 29 mutation in intron I of the hepatic lipase gene 10719 Berlin (DE) leads to alternative splicing resulting in enzyme deficiency",JOURNAL OF LIPIDRESEARCH, vol. (56) References cited: 37, no. 6, 1996, pages 1213-1223, XP002678284, EP-A2- 1 516 929 WO-A1-93/03151 ISSN: 0022-2275 WO-A1-2009/049889 WO-A2-2008/005675 • Palani Kumaresan ET AL: "High-Throughput WO-A2-2008/104184 WO-A2-2012/042374 Single Copy DNA Amplification and Cell Analysis in Engineered Nanoliter Droplets", Analytical Chemistry, vol. 80, no. 10, 1 May 2008 (2008-05-01), pages 3522-3529, XP055019414, ISSN: 0003-2700, DOI: 10.1021/ac800327d Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 626 433 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 626 433 B1 • SUMATHI RAMACHANDRAN ET AL: ’Evaluation of Intra-Host Variants of the Entire Hepatitis B Virus Genome’ PLOS ONE vol. 6, no. 9, E25232, 20 September 2011, pages 1 - 10, XP055099652 DOI: 10.1371/journal.pone.0025232 2 EP 2 626 433 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention is in the field of molecular biology, diagnostics and more in particular also medicine. It is also in the field of biochemistry and molecular biology. BACKGROUND 10 [0002] It is difficult to determine the degree of variation on the level of single cells in a heterogeneous cell population for single-nucleotide polymorphisms (SNPs), variable sequence regions and splice variants. If bulk nucleic acids are isolated from cell populations, the information which nucleic acid variants were present in which combination in each individual cell is lost. [0003] This information can be important in cases where different nucleic acid variants act together in cells to determine 15 the specific biological behavior of the cells. For example, two mutations in two different signaling pathway molecules can result in malignancy of individual cancer cells, whereas other cells in the same tumor population that carry only one of these mutations are non-malignant. Other examples include the variable T cell receptor alpha and beta chain genes and transcripts, which act together in each T cell to produce T cell receptors that are variable among each T cell, and the immunoglobulin variable heavy chain (VH) and variable light chain (VL) genes and transcripts present in B cells, 20 which act together in each B cell to produce immunoglobulins that are variable among each B cell. [0004] Conventionally, combinations of nucleic acid variations can be analyzed after isolating single cells through simple titration, through cell picking or through fluorescence-activated cells sorting (FACS). Subsequently, the combi- nations of sequence variants can be analyzed after amplification of the nucleic acids of interest from each individual cell by polymerase chain reaction (PCR) or reverse transcription-PCR (RT-PCR). Analysis methods include nucleic acid 25 sequencing, hybridization on microarrays or quantitative real-time PCR (qPCR). In order to facilitate the analysis of the pairing of nucleic acid variations, different gene sequences or reverse-transcribed RNA sequences can be coupled by overlap PCR, which has been reported to be compatible with water-in-oil emulsions. A method for coupling variable regions of immunoglobulin genes by multiplex overlap-extension RT-PCR from isolated single cells has been described before (US7749697B2). Similar methods have been used by others to clone functional antibody variable domains in the 30 form of single-chain variable fragments (scFv) from natural repertoires such as hybridoma cells and spleen cells. [0005] Another method for coupling of two or more copies of non-contiguous DNA sequences from single cells of a heterogeneous population has been described before (Patent EP597960B1). Gene elements can be coupled inside single cells (in situ "in-cell PCR") within intact or substantially intact cells after cell fixing e.g. with formaldehyde and subsequent cell permeabilization to ensure access of PCR reagents to the gene elements (see WO2008/104184 & 35 Embleton MJ, Gorochov G, Jones PT, Winter G. In-cell PCR from mRNA: amplifying and linking the rearranged immu- noglobulin heavy and light chain V-genes within single cells. Nucleic Acids Res. 1992 Aug 11; 20(15):3831-7). Alterna- tively, cells and PCR reagents can be introduced into aqueous droplets and dispersed in an organic phase as an emulsion, wherein each droplet contains preferentially only one cell, such that gene elements from single cells are coupled together. [0006] The methods however are tedious. 40 [0007] The technical problem underlying the present invention is the provision of an enhanced method that facilitates the analysis of nucleic acid molecules in cases where these molecules act together in a cell. [0008] The technical problem is solved by the embodiments provided herein. The invention is defined by the claims. DEFINITIONS 45 [0009] Herein, a "sample" is a sample of bodily fluid, e.g. from a patient which is to be subjected to the herein disclosed method, for example to be diagnosed. Such samples may be from a human, a plant or an animal. Preferred samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions. In addition, one of skill in the art would realize that some test samples would be more readily analyzed following a fractionation or purification 50 procedure, for example, separation of whole blood into serum or plasma components. [0010] Thus, the sample is selected from the group comprising a blood sample, a serum sample, a plasma sample, a cerebrospinal fluid sample, a saliva sample and a urine sample or an extract of any of the aforementioned samples. Preferably, the sample is a blood sample, most preferably a serum sample or a plasma sample. The sample may include tissue, a cell, a bacterium and/or a virus. 55 [0011] Herein "tissue" is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function (blood, liver etc.). [0012] Herein a "fraction thereof" or a "fraction from a sample" is any part of the sample such as a part of the tissue, 3 EP 2 626 433 B1 a part of the cell(s), a part of the bacteria and/or viruses that can be isolated in a reproducible manner and has been enriched for a specified subject manner with the tissue, cell(s), bacteria and/or viruses. Typical examples are exosomes, a cellular structure, a sub-cellular compartment, and/or organelle fractions. [0013] Herein "emulsions" are thermodynamically stable, isotropic liquid mixtures of oil, water and surfactant, frequently 5 in combination with a cosurfactant. The aqueous phase may contain salt(s) and/or other ingredients, and the "oil" may actually be a complex mixture of different hydrocarbons and olefins. In contrast to ordinary emulsions, microemulsions form upon simple mixing of the components and do not require the high shear conditions generally used in the formation of ordinary emulsions. The three basic types of microemulsions are direct (oil dispersed in water, o/w), reversed (water dispersed in oil, w/o) and bicontinuous. In ternary systems such as microemulsions, where two immiscible 10 phases (water and ’oil’) are present with a surfactant, the surfactant molecules may form a monolayer at the interface between the oil and water, with the hydrophobic tails of the surfactant molecules dissolved in the oil phase and the hydrophilic head groups in the aqueous phase.