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(12) United States Patent (10) Patent No.: US 8,663,919 B2 Dong Et Al USOO8663919B2 (12) United States Patent (10) Patent No.: US 8,663,919 B2 Dong et al. (45) Date of Patent: Mar. 4, 2014 (54) CHROMOSOME CONFORMATION (56) References Cited ANALYSIS U.S. PATENT DOCUMENTS (75) Inventors: Shoulian Dong, Mountain View, CA 2005/OO32105 A1* 2/2005 Bair et al. ......................... 435/6 (US); Junko F. Stevens, Menlo Park, CA 2009/0061425 A1* 3/2009 Lo et al. ............................ 435/6 (US); Chunmei Liu, Palo Alto, CA 2010.0081141 A1 4/2010 Chen (US); Cora L. Woo, Santa Clara, CA (US); Luz Montesclaros, Pittsburg, CA FOREIGN PATENT DOCUMENTS (US) EP 1835.038 9, 2007 Assignee: Life Technologies Corporation, EP 2083090 T 2009 (73) WO 2009,147386 12/2009 Carlsbad, CA (US) WO 2011, 146056 11, 2011 (*) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. Berensmeier (2006) “Magnetic particles for the separation and purificationofnucleic acids' Appl Microbiol Biotechnol 73:495-504; Appl. No.: 13/475,089 published online Oct. 25, 2006.* (21) Hagege, Helene et al., “Quantitative Analysis of Chromosome Con Filed: May 18, 2012 formation Capture Assays (3C-qPCR)', Nature Protocols, vol. 2, No. (22) 7, 2007, 1722-1733. Partial International Search Report for International Application No. (65) Prior Publication Data PCT/US2012/038558 dated Sep. 24, 2012. US 2012/O3O2449 A1 Nov. 29, 2012 International Search Report and Written Opinion for International Appl. No. PCT/US2012/03855 mailed Jan. 15, 2013. Dekker, Job et al., "Capturing Chromosome Conformation'. Science, Related U.S. Application Data vol. 295, 2002, 1306-1311. (60) Provisional application No. 61/487.614, filed on May * cited by examiner 18, 2011, provisional application No. 61/579,444, filed on Dec. 22, 2011, provisional application No. Primary Examiner — Christopher M Babic 61/579,876, filed on Dec. 23, 2011. Assistant Examiner — Karen S Weiler (51) Int. C. (57) ABSTRACT CI2O I/68 (2006.01) Disclosed herein are compositions, methods and kits for ana (52) U.S. C. lyzing three-dimensional chromatin and/or chromosome USPC ........................................................... 435/6.1 conformation. Method are also disclosed for using the meth (58) Field of Classification Search ods disclosed herein for diagnosing diseases such as cancer. None See application file for complete search history. 7 Claims, 27 Drawing Sheets U.S. Patent Mar. 4, 2014 Sheet 1 of 27 US 8,663,919 B2 o U.S. Patent Mar. 4, 2014 Sheet 3 of 27 US 8,663,919 B2 U.S. Patent Mar. 4, 2014 Sheet 4 of 27 US 8,663,919 B2 U.S. Patent Mar. 4, 2014 Sheet 5 Of 27 US 8,663,919 B2 U.S. Patent US 8,663,919 B2 U.S. Patent US 8,663,919 B2 U.S. Patent US 8,663,919 B2 U.S. Patent Mar. 4, 2014 Sheet 10 of 27 US 8,663,919 B2 90 ??. 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U.S. Patent Mar. 4, 2014 Sheet 16 of 27 US 8,663,919 B2 Amplification Piot Amplification Piot O3 + 1,000 E-3 di:. 1.000E-4 di. Detector. 8sw Fio: A R vs. joie Threshoic:50 F.G. 5A U.S. Patent Mar. 4, 2014 Sheet 17 Of 27 US 8,663,919 B2 3} 250 20 50 O 5 interra adaptor incorporated targets G. 53 U.S. Patent Mar. 4, 2014 Sheet 18 of 27 US 8,663,919 B2 - - - - F.G. 6A U.S. Patent Mar. 4, 2014 Sheet 19 Of 27 US 8,663,919 B2 80 - 80 - 45w 35 30 250 - 20 5. Sr. F.G. 6B U.S. Patent Mar. 4, 2014 Sheet 20 Of 27 US 8,663,919 B2 uô?søgog U.S. Patent Mar. 4, 2014 Sheet 21 of 27 US 8,663,919 B2 $3',d???šan N-O bo e 80 U.S. Patent Mar. 4, 2014 Sheet 22 of 27 US 8,663,919 B2 ...r.l...r...r.l...r...r.l...r....r. seeeeeeeee N-O & O eja Å U.S. Patent Mar. 4, 2014 Sheet 23 Of 27 US 8,663,919 B2 Aligned Met Curves 5 66 67 6869 to 7, 72 7374 is 76 77 is 7e 80 81628384 85 36 8788 emperature {C} A: 0% Methyl, Cr? at 23 B: 100% Methyl FG 9A U.S. Patent Mar. 4, 2014 Sheet 24 of 27 US 8,663,919 B2 PD Expression 23 Confo frn GA / MCF-7 Control not in GAPDH F.G. 93 U.S. Patent Mar. 4, 2014 Sheet 25 Of 27 US 8,663,919 B2 W six-sc on Oo - Co is a ri () c- Y - O - c < *; k (- - - - x---see % S85te 3 po U.S. Patent Mar. 4, 2014 Sheet 26 of 27 US 8,663,919 B2 r s s S.- S.- SNNNN, NY, i. s res NNNicx, NNNNNNNNN, CNN st s--- Sefuel O pio U.S. Patent Mar. 4, 2014 Sheet 27 Of 27 US 8,663,919 B2 BC2 2. 20.5 20.0 9.5 - - BC2 Expression. & AC-7 231st 5 9.0 8.5 8,0-- --------------------------------------- 17.5 23 Contro MCF-7 Control FG. 21A hsa-MiR-2 Expression 28,5- 28.35 - 28 AiR 21 Expression: 27.5 C7|3:5 27 26.5 25.5- 24.5 23 Contro Nor MCF Control Nor FG 21B US 8,663,919 B2 1. 2 CHROMOSOME CONFORMATION ization (FISH) analysis can examine multiple loci, but the ANALYSIS severe experimental conditions may adversely affect chromo Somal organization. CROSS-REFERENCE TO RELATED In an attempt to overcome the limitations of visual chro APPLICATIONS mosomal analysis, methods using the polymerase chain reac tion (PCR) have been developed. For example, the chromo This application claims priority to U.S. Provisional Appli Some conformation capture (3C) method analyzes overall cation No. 61/487,614, filed May 18, 2011, U.S. Provisional chromosomal spatial organization and physical properties at Application No. 61/579,444, filed Dec. 22, 2011 and U.S. a higher resolution (see, Dekker et al., Science 295:1306 Provisional Application No. 61/579,876, filed Dec. 23, 2011 10 1311 (2002)). In3C experiments, genomic DNA (gDNA) and proteins in the chromosomes are fixed in place by cross which disclosures are herein incorporated by reference in linking. The cross-linked gldNA is digested by a restriction their entirety. enzyme and ligated before being purified for analysis. Physi FIELD cal interactions between genomic loci are identified as spe 15 cific cross-ligated DNA elements using PCR amplification. As a result of 3C methods, spatial information is converted to This disclosure relates to methods, reagents and kits useful quantifiable DNA sequences. However, the wide adaptation for epigenetic analysis of DNA. of 3C methods has been hindered by the lack of quantitative process controls and cumbersome protocols. BACKGROUND Derivative methods of 3C, such as 4C and 5C, have also been developed. 4C and 5C differ from 3C only in the analysis Epigenetics is the study of heritable changes in gene func of the ligation product. In 4C, the ligation product is first tion that do not involve changes in DNA sequence. These amplified by PCR using two outwardly facing primers from changes can occur due to the chemical modification of spe the restriction site to create a circular DNA molecule which is cific genes or gene-associated proteins of an organism. These 25 then analyzed by microarray technology. In 5C, the ligation modifications can affect how genes are expressed and used in products are mixed with special primers designed to anneal at cells. For example, methylation of specific regions in gene the ends of the restriction fragment. Analysis is carried out sequences. Such as CpG sites, can make these genes less either by use of a microarray or by sequencing against a 3C transcriptionally active. Another example is post-transla library of ligation products. tional modification of histone proteins around which genomic 30 However, there are several disadvantages associated with DNA is wound. This histone modification can affect the these 3C-based methods. The assays are time consuming and unwinding of the DNA during transcription which, in turn, are not precise and sensitive enough for reactions with low can affect the expression of the transcribed genes. Further digestion or ligation efficiency. These methods also require more, chromosome conformation or chromatin compaction significant dilution of the DNA sample. As a result, large can also have an effect on gene expression, Such as affecting 35 quantities of the sample may be needed, which may not the accessibility of the template to polymerases or changing always be available.
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