US010422001B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 ,422 , 001 B2 Van Eijk et al. ( 45 ) Date of Patent: " Sep . 24 , 2019 ( 54 ) SEQUENCE BASED GENOTYPING BASED 2005/ 0239089 A1 * 10 / 2005 Johnson .. C12Q 1 /6827 435 /6 . 12 ON OLIGONUCLEOTIDE LIGATION ASSAYS 2006 / 0211000 A19 /2006 Sorge et al . 2007 /0196849 A1 * 8 /2007 Spier .. .. .. .. C12Q 1 /6827 (71 ) Applicant: Keygene N . V ., Wageningen (NL ) 435 /6 . 16 (72 ) Inventors : Michael Josephus Theresia Van Eijk , Wageningen (NL ) ; René Cornelis FOREIGN PATENT DOCUMENTS Josephus Hogers , Wageningen (NL ) EP 0 185 494 6 / 1986 EP 0 246 864 B1 11 / 1987 ( 73 ) Assignee : KEYGENE N . V ., Wageningen (NL ) EP 0 745 140 12 / 1996 EP 0912 761 5 / 1999 EP 0 956 359 11 / 1999 ( * ) Notice : Subject to any disclaimer , the term of this EP 0 964 704 12 / 1999 patent is extended or adjusted under 35 EP 1 194 770 4 / 2002 U . S . C . 154 ( b ) by 0 days. EP 1 252 334 10 / 2002 EP 1 255 871 11 / 2002 This patent is subject to a terminal dis EP 1 313 880 5 / 2003 claimer . WO WO - 96 / 15271 5 / 1996 WO WO - 97 / 45559 12 / 1997 WO WO - 98 / 04745 2 / 1998 ( 21 ) Appl . No. : 15/ 683 , 063 WO WO - 98 / 04746 2 / 1998 WO WO - 03 /033722 A2 4 / 2003 ( 22 ) Filed : Aug. 22 , 2017 WO WO - 03 /052142 6 / 2003 WO WO - 03 /054511 7 / 2003 (65 ) Prior Publication Data WO WO - 03 /060163 A2 7 / 2003 WO WO - 2004 / 111271 12 /2004 US 2018 /0023135 A1 Jan . 25 , 2018 WO WO - 2005 /021794 3 / 2005 WO WO - 2005 /026389 A2 3 / 2005 Po wo WO - 2005 / 118847 12 / 2005 Related U . S . Application Data WO WO - 2007 /073165 6 / 2007 Continuation of application No. 14 / 131 , 181 , filed as WO W O - 2007 / 100243 AL 9 / 2007 application No . PCT/ NL2012 / 050493 on Jul . 9 , 2012 , now Pat . No . 9 ,777 ,322 . OTHER PUBLICATIONS Deschamps, et al. “ Utilization of next- generation sequencing plat (60 ) Provisional application No . 61/ 505 ,787 , filed on Jul. forms in plant genomics and genetic variant discovery ” , Mol 8 , 2011 . Breeding ( 2010 ) vol. 25 , pp . 553 - 570 . (51 ) Int. Ci. International Search Report received in PCT/ NL2012 /050493 dated Oct . 2 , 2012 . C120 1 /6874 ( 2018 .01 ) Nilsson , et al . “ Padlock Probes : Circularizing Oligonucleotides for C12Q 1 /6827 ( 2018 .01 ) Localized DNA Detection ” , Science , 1994 , vol. 265 , pp . 2085 - 2088 . C12Q 1 /683 ( 2018 .01 ) Nilsson , et al ., “ Making Ends Meet in Genetic Analysis Using (52 ) U .S . CI. Padlock Probes ” , Human Mutation , 2002 , vol . 19 , pp . 410 - 415 . CPC .. .. C12Q 1 /6874 ( 2013 .01 ) ; C12Q 1 /683 Van Eijk , et al ., “ SNPWave TM : a flexible multiplexed SNP ( 2013 .01 ) ; C12Q 1 /6827 ( 2013 . 01 ) genotyping technology ” , Nucleic Acids Research , 2004 (published ( 58 ) Field of Classification Search online Mar . 5 , 2004 ) , vol. 32 , No. 4 , p . e47 . CPC . .. C12Q 1 /6811 ; C12Q 1 /6874 See application file for complete search history . * cited by examiner Primary Examiner — Narayan K Bhat ( 56 ) References Cited (74 ) Attorney , Agent, or Firm — Foley & Lardner LLP U . S . PATENT DOCUMENTS (57 ) ABSTRACT 4 , 988 ,617 A 1 / 1991 Landegren et al. The invention relates to a method for the detection of a target 5 , 470 ,705 A 11/ 1995 Grossman et al. nucleotide sequence in a sample based on an oligonucleotide 5 ,521 , 065 A 5 / 1996 Whiteley et al . ligation assay wherein probes are used that contain ( a 5 , 876 , 924 A 3 / 1999 Zhang et al . combination of) sequence -based identifiers that can identify 5 , 962 , 223 A 10 / 1999 Whiteley et al. the sample and the target sequence ( i . e . locus and /or allele 6 , 027 , 889 A 2 / 2000 Barany et al. 6 , 156 , 178 A 12 / 2000 Mansfield et al . combination ) wherein after the ligation step , the ligated 6 , 221, 603 B 4 / 2001 Mahtani probes, or after amplification , the amplified ligated probes , 8 , 460 ,866 B2 6 / 2013 Van Eijk et al . are restricted using restriction enzymes to cut of part of the 2003 /0032016 A12 / 2003 Barany et al . probes and continue with those parts ( identifiers and target 2003 /0108913 A1 * 6 / 2003 Schouten C12Q 1 /686 sequence ) that contain the relevant information in the 435 /6 . 18 sequencing step . 2003 /0119004 Al 6 / 2003 Wenz et al. 2003 / 0190646 A1 10 / 2003 Wenz et al . 14 Claims, 6 Drawing Sheets U . S . Patent Sep. 24 , 2019 Sheet 1 of 6 US 10 ,422 ,001 B2 PBS2PBS2 ID2 RE2 PBS2|1PR2 ID2 RE2 PBS11PR2 T T TAG2TAG2 PR2 TAG2 - TAG2 TS2 P2P2 TS2 POP2 TS2 P2 1FIG LLLLLLLLLLLLLLLLLL TS1 TS P1 ID1TS1 TAG1 APR1on TAG1 MMMM ITAGI HAPRI PT wwwwwwwwwwwwwwwwwwwwwwwwtwintatata??????????????????????????????????????????????* PBSIHAPRAPA PBS1|TAG1 101 RE1 REI PBS1 5 5 FIG1A FIG1B FIG1C U . S . Patent Sep. 24 , 2019 Sheet 2 of 6 US 10 ,422 ,001 B2 FIG 2 RE1 TS1 * * TS2 RE2 TAG1 PBS1 TAG2 PBS2 p1 P2 Hybridization /Ligation Restriction Amplification VIII IIIIIIIIIIIII ???????????????? P2 RLP 1 anananananananananananananan Restriction ananananananananananan RA Sequencing Identification AD2D1 AD1 D1 FIG 2A 1. JADI AD2 U . S . Patent Sep. 24 , 2019 Sheet 3 of 6 US 10 ,422 ,001 B2 FIG 3 OOOOO | TS 1D RE | PBS1YANNN | PBS2AD1AD2N NN PR1 PR21 RLPIRA FIG 3A PBS1 REI 1D1 TS1 TS2 D2 RE2 PBS2 MILIMW5 LOCUS PR1 PR2 FIG 3B : : : : : : - . V VIRUZZOLI39: 1110 WW ZIALI INIZA 2 my ! . A RBUD CIATI KANAZDA 3 VIXITX . CIA 2 L IZZZZZZZZZZZ4 w 1 WM TILLATIVIURD 6 : : : : : FA WAISLITZZZZZXIZMA3. 7 HOHOHOHOHOHOU 1 . 1 . RE . 1 . 3 : . Wow 129900W UELE . 33399UNUIX273337 / . : . : . : . : . : . : . S . : : : . " 09 : 30H . W PA N U . S . Patent Sep. 24 , 2019 Sheet 4 of 6 US 10 ,422 ,001 B2 FIG 4 FIG 4A 2 : : : : : : : : : : : : :: : B 3 3333333 Recherchede DNDODONO Demburu w wwhhhhhhhhhhhhhhhhhhhhhhhhhhhhhmmmmDDDDDDDDDDDDDDDDDDDDDDD D DD M M M on W K OM O -M M- - -Momo - XX FIG 4B ANXHXH * . * . UNTV L : : : : : : : : : : DOOOOOOOOOOOOOO P J Whorn0900333 here are more and more minu minu meelest om man in 999 Long - - - - CA Short mXOXOBE - - - - - FIG 4C EPHEM . .PONOVOVODNO OKOVOVODIONONONONONDUCONONONOHODNOKONONOODLOOKOOKOONPANONONONONNONDONONONONONDUCONONONONONDONU! Long 1 - - - - - - * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * OVODOVODOVODOVODOVODOVODOVODOVODOVOVODKOVODKOVODNOTOVOVOUOVOVOUOVOVOUOVOVOVODVOUHOVOVOVOUOVOVO . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * EMVILLE Long 2 - i - - -te - -to oO FIG 4D wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww . : : : : : : : DIDINDIDIDKADINDADIRDIDONADONDADADADADADADADADIONIDIOIOIOIOIOIDIDINDINIDIDUNDIDADADADADADADADADADAD: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : . S A E . SE Long 1 - - - - Short YAYINYWY - - - Long 2 U . S . Patent Sep. 24 , 2019 Sheet 5 of 6 US 10, 422 , 001 B2 FIG 5A ?? ? ** * * * ..* . ?? ** * * * * * ?? 93 as - ??? ?????? ???? ????????????????? ???? ???????????????????????????????????????? ???? ?? " " : " : " " " " " " ?? " : ' : ' : ' : ' : ' 12 . 0 + U . S . Patent Sep. 24 , 2019 Sheet 6 of 6 US 10 ,422 ,001 B2 FIG 6 PBS1 RE1 101 . TS1 TS2 D2 RE2 PBS244444444 II Amplification mm Circularise . .. Shear | Adapter ligation HILLI . ' ' . ' . ' . US 10 , 422 , 001 B2 SEQUENCE BASED GENOTYPING BASED nucleotides are selected to anneal to immediately adjacent ON OLIGONUCLEOTIDE LIGATION ASSAYS segments of the sequence to be determined . One of the selected oligonucleotide probes has an end region wherein CROSS -REFERENCE TO RELATED one of the end region nucleotides is complementary to either APPLICATIONS 5 the normal or to the mutated nucleotide at the corresponding position in the known nucleic acid sequence . A ligase is This application is a Continuation of U . S . patent appli provided which covalently connects the two probes when cation Ser . No . 14 / 131, 181 , filed Apr. 17 , 2014 , which is the they are correctly base paired and are located immediately U . S . National Phase of International Patent Application No . adjacent to each other . The presence , absence or amount of PCT /NL2012 /050493 , filed Jul. 9 , 2012 , published on Jan . 10 the linked probes is an indication of the presence of the 17 , 2013 as WO 2013 /009175 A1, which claims priority to known sequencesequence andand / or mutation .. Other variantsvari of OLA U . S . Provisional Application No . 61/ 505 , 787, filed Jul. 8 , based techniques have been disclosed inter alia in Nilsson et 2011 . The contents of these applications are herein incor al. Human mutation , 2002 , 19 , 410 -415 ; Science 1994 , 265 : porated by reference in their entirety . 2085 - 2088 ; U . S . Pat. No . 5 , 876 , 924 ; WO 98 / 04745 ; WO 15 98 /04746 ; U . S . Pat. Nos . 6 , 221 ,603 ; 5 ,521 , 065; 5 , 962 ,223 ; FIELD OF THE INVENTION EP 185494BI; U . S . Pat . Nos. 6 , 027 ,889 ; 4 , 988 ,617 ; EP 24686481 ; U . S . Pat . No. 6 , 156 , 178 ; EP 745140 BI; EP The present invention relates to the field of molecular 964704 BI; WO 03 /054511 ; US 2003 /0119004 ; US 2003 / biology and biotechnology . In particular the invention 190646 ; EP 1313880 ; US2003 / 0032016 ; EP 912761; EP relates to the field of nucleic acid detection , more in par - 20 956359 ; US 2003 / 108913 ; EP 1255871 ; EP 1194770 ; EP ticular to the design and composition of ( collections ) of 1252334 ; W096 / 15271 ; W097 / 45559 ; US2003 / probes that can be used for the high throughput detection of 0119004A1; U . S . Pat. No . 5 ,470 ,705 . nucleic acids. The invention also relates to methods for the Further advancements in the OLA