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(12) United States Patent (10) Patent No.: US 8,673,560 B2 Leamon Et Al

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(12) United States Patent (10) Patent No.: US 8,673,560 B2 Leamon Et Al USOO867356OB2 (12) United States Patent (10) Patent No.: US 8,673,560 B2 LeamOn et al. (45) Date of Patent: *Mar. 18, 2014 (54) METHODS AND COMPOSITIONS FOR (51) Int. Cl. MULTIPLEX PCR CI2O I/68 (2006.01) CI2P 19/34 (2006.01) (75) Inventors: John Leamon, Guilford, CT (US); C07H 2 1/02 (2006.01) Mark Andersen, Carlsbad, CA (US); CO7H21/04 (2006.01) Michael Thornton, San Francisco, CA (52) U.S. Cl. (US) USPC .......... 435/6.1; 435/91.2:536/23.1; 536/24.3 (58) Field of Classification Search (73) Assignee: Life Technologies Corporation, None Carlsbad, CA (US) See application file for complete search history. (*) Notice: Subject to any disclaimer, the term of this (56) References Cited patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. U.S. PATENT DOCUMENTS This patent is Subject to a terminal dis 5,582,989 A 12/1996 Caskey et al. claimer. 5,814,491 A 9/1998 Viget al. 5,882,856 A 3, 1999 Shuber et al. (21) Appl. No.: 13/618,805 (Continued) (22) Filed: Sep. 14, 2012 OTHER PUBLICATIONS (65) Prior Publication Data Stiller, M. et al., Genome Res., vol. 19, pp. 1843-1848 (2009).* US 2013/OO11881 A1 Jan. 10, 2013 (Continued) Related U.S. Application Data Primary Examiner — Teresa E Strzelecka (63) Continuation of application No. 13/458,739, filed on (57) ABSTRACT Apr. 27, 2012. The present invention provides methods, compositions, kits, (60) Provisional application No. 61/479,952, filed on Apr. systems and apparatus that are useful for multiplex PCR of 28, 2011, provisional application No. 61/531,583, one or more nucleic acids present in a sample. In particular, filed on Sep. 6, 2011, provisional application No. various target-specific primers are provided that allow for the 61/531,574, filed on Sep. 6, 2011, provisional selective amplification of one or more target sequences. In application No. 61/538,079, filed on Sep. 22, 2011, one aspect, the invention relates to target-specific primers provisional application No. 61/564,763, filed on Nov. useful for the selective amplification of one or more target 29, 2011, provisional application No. 61/578,192, sequences associated with cancer or inherited disease. In filed on Dec. 20, 2011, provisional application No. Some aspects, amplified target sequences obtained using the 61/594,160, filed on Feb. 2, 2012, provisional disclosed methods, kits, systems and apparatuses can be used application No. 61/598.881, filed on Feb. 14, 2012, in various downstream processes including nucleic acid provisional application No. 61/598,892, filed on Feb. sequencing and used to detect the presence of genetic vari 14, 2012, provisional application No. 61/625,596, antS. filed on Apr. 17, 2012, provisional application No. 61/639,017, filed on Apr. 26, 2012. 9 Claims, 53 Drawing Sheets &f by Exon-Specific ar, R: 45&p$435 id-65 finer Reinows y and Repair : RetroveAutiglex 9.XX 38YXSP 8 NP : Primers .igation : Attach 42% Uruversal fig’? Adacts US 8,673,560 B2 Page 2 (56) References Cited PCT/US2012/035612. International Search Report and Written Opinion mailed on Jul. 25, 2012. U.S. PATENT DOCUMENTS Varley, Katherine et al., “Nested Patch PCR enables highly multi plexed mutation discovery in candidate genes'. Genome Res., 18. 6,207,372 B1 3, 2001 Shuber 2008, 1844-1850. 6,251,588 B1 6/2001 Shannon et al. PCT/US2012/062494, International Search Report and Written 7,101,663 B2 9/2006 Godfrey et al. Opinion mailed on Jan. 17, 2013, 1-11. 7,118,867 B2 10/2006 Tabiti et al. 7,217,515 B2 5, 2007 Chiu et al. PCT/US2012/065650, International Search Report and Written 7,670,774 B2 3/2010 Moon et al. Opinion mailed on Jan. 23, 2013, 1-12. 7,687,247 B1 3/2010 Hartley et al. Stiller, Mathias et al., “Direct multiplex sequencing (DMPS)—a 2008/0228589 A1 9, 2008 Koehler et al. novel method for targeted high-throughput sequencing of ancient and 2008/029.3589 A1 1 1/2008 Shapero highly degraded DNA”. Genome Research, Supplemental Material, 2010/0129874 A1 5, 2010 Mitra et al. 2009, 1-15. 2011/0212490 A1 9/2011 Fredriksson et al. Meyer, Matthias et al., “Illumina Sequencing Library Preparation for 2011/0319290 A1* 12/2011 Raymond et al. ................. 506.9 Highly Multiplexed Target Capture and Sequencing'. Cold Spring Harbor Protocols, vol. 2010, protocolS448, 2010, 1-10. OTHER PUBLICATIONS Buck, et al., “Design strategies and performance of custom sequenc ing primers'. BioTechniques, 27, 1999, 528-536. Longo, M.C. et al., Gene, vol. 93, pp. 125-128 (1990).* Dieffenbach, C.W. et al., “General concepts for PCR primer design”. Cross, Deanna et al., “Development of a fingerprinting panel using Genome Res., 3:, 1993, S30-S37. medically relevant polymorphisms”. BMC Medical Genomics, 2:17. http://www.biomedcentral.com/1755-8794/2/17, 2009. * cited by examiner U.S. Patent Mar. 18, 2014 Sheet 1 of 53 US 8,673,560 B2 Forward For Wafd 5 -----------Air XXX XXXSpecif ** N 3. N odified Basess - AN 3 8-8-8-8-8-8 5 GeneReverse Specific ReverseAmp FG. A DDDDDD (SODDDDD. F.G. C. 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