USOO5876942A United States Patent (19) 11 Patent Number: 5,876,942 Cheng et al. (45) Date of Patent: Mar. 2, 1999 54 PROCESS FOR SEXING COW EMBRYOS Gibson et al., Biochemistry 30:1075–1079 (1991). 75 Inventors: Winston Teng-Kuei Cheng, Taipei; Shimokawa et al., J. of Biological Chemistry 262(9) : Chuan-Mu Chen, Tai Chung; Che-Lin 4042–4047 (1987). Hu, Taipei; Chih-Hua Wang, Taipei; Schafer et al., BioEssays 18(12):955-962 (1996). Kong-Bung Choo, Taipei, all of Taiwan Sullivan et al., BioTechniques 15(4):636-641 (1993). 73 Assignee: National Science Council of Republic Ennis et al., Animal Genetics 25:425-427 (1994). of China, Taipei, Taiwan Primary Examiner W. Gary Jones 21 Appl. No.: 899,811 ASSistant Examiner Ethan Whisenant Attorney, Agent, or Firm-Bacon & Thomas 22 Filed: Jul. 24, 1997 57 ABSTRACT 51) Int. Cl. ............................. C12Q 1/68; CO7H 21/02; CO7H 21/04 A rapid, highly reproducible and Sensitive technique has 52 U.S. Cl. ............................... 435/6; 536/231; 536/24.3 been Successfully developed for Sexing the cow embryos, by 58 Field of Search ................................ 435/6; 536/23.1, method of polymerase chain reaction (PCR) against the 536/24.3;985/77, 78 amelogenin (bAML) genes located on both X- and Y-chromosomes of the Holstein dairy cattle. Results from 56) References Cited DNA sequence analysis showed that there was only 45% homology between the intron 5 of AMLX and AMLY genes. U.S. PATENT DOCUMENTS Based on these Sequences a pair of Sex-Specific primers, 5,578,449 11/1996 Frasch et al. ............................... 435/6 pbAML5XY(+) and pbAML5XY (-),were designed allow 5,596,089 1/1997 Silversides et al. ................... 536/24.3 ing to amplify a single fragment of 476-bp from the female cattle and two fragments of 476-bp and 341-bp from the OTHER PUBLICATIONS male ones, respectively. The most important feature is that Sommer et al., Nucleic Acids Research 16(6): 6749 (1989). the precise Sensitivity of Sex-determination was confirmed to Kirkpatrick et al., J. of Reproduction and Fertility 98 : be reached as minimum template as trace amount of 335-340 (1993). genomic DNA content in either a single lymphocyte or a Pompet al., J. Animal Science 73 : 1408–1415 (1995). Single blastomere isolated from cow embryo at day-6 to Aasen et al., BioTechnology 8: 1279–1281 (1990). day-7. Moreover, neither those of complicated procedures Darling et al., PNAS 83:135-139 (1986). for purifying the DNA prior the PCR nor any extra pair of Gibson et al., Biochemistry 31(35): 8384–8388 (1992). primers for Serving as internal control is thought to be Gibson et al., Biochemical and Biophysical Research Com essential and the Sex-determination of over hundred munications 174(3): 1306–1312 (1991). embryos can be completed at once within 4hrs. Suzumori et al., Obstetrics & Gynecology 80(1): 150-154 (1992). 7 Claims, 13 Drawing Sheets :::::::::::: 88: U.S. Patent Mar. 2, 1999 Sheet 1 of 13 5,876,942 U.S. Patent Mar. 2, 1999 Sheet 2 of 13 5,876,942 U.S. Patent Mar. 2, 1999 Sheet 3 of 13 5,876,942 LOCUS BOVALY 1539 bp dis-DNA MAN 28-0CT-1995 DEFINITION Bos taurus chronosoine Y a melogenin (AMLY) gene, complete intron 5. KEYWORDSACCESSION anielL.48609 Ogenin. SOURCE Bos taurus (strain Holstein) adult blood DNA. ORGANISM Bos taurus Eukaryota; Animalia; Chordata; Vertebrata; Mammalia; Theria; Elia; Artiodactyla; Ruminantia; Pecora; Bovidae; Bovinae; OWll. REFERENCE 1 (bases 1 to 1533) TITLEAUTHORS ChengCheng, paperW.F.K., Chen, C.-M., Hu, C.-L., Wang, C.-H. and Choo, K.-B. JOURNA Unpublished (1995) STANDARD full staff review REFERENCE 2 (sites) AUTHORSTITLE (No ni.titlel ORA inpublished (1995) STANDARD full staff review FEATURES Location/Qualifiers itron ... 1539 /nusber=5 /ap=" chronoso Re Y" /gene="AMLY. ?evidence=EXPERIMENTAL /citation={2} SOCe ... 1539 forganis "Bos taurus" Wstrain="Holstein" /cell type=leukocyte" /dev stage="adult" /germline /macronuclear /sequenced aol="DNA" /tissue types"blood" BASE COUNT 544 a 26 C 245 g 534 t ORIGIN l gtaagtacaC Ctta aggcCa ttacaa.ca.g titatgaaaat aat C2agCaa aaatagcCCa 6. Cagaaat Caa aattctctica CagtCCaaga CCtagagttct CaaCtgCagt tagtgattact 121 attattocCaia gtgttgttgttg taagtttata aatgagCtta tittatctata taggcatacac 181 tttgttgaatt CaaaatctaC aattatttgg aattittaatg gCalatatgala tgCtattga 24 aatgcagtat aataaaggca aCaatctta tictt Ctggitt gCttaactag taCattagaC 301 ttata Cagala ataaataaaa. agagtattt C tgggattgag gagalaggaag tatCagttitt 361. aaaaaattat agctggat.ct tCaaggCtct aaagaaatat taCCtgttgt aggtgattitt 421 aaagtaattit ggaalacagtt. aaagtagaC8 aacgtagatt Ctatt Catt CCCaggatta 48 aaaaaaattit taataagaat gttitCataac atagcttaaa atticCCCC Cact tcagaa 541 agcttittggag gaataattga gaagitaa CCC aataagggtC aCCttCCatt Catt CCtgta 6Cl aaCCCtcatt tCtctgcatt aagtt tagta attcta Caat taataaatgC tgttagagat 661 ttgctacga. gaaaaCaCtg aagagtaagg tagaaataac Ctagaatatat agtCaggata 72 tgttggCttct attgCtagct CtgCCCtgaa gaagaaattit a Cagctgttt tCct Ctaaat 78. gtttCCtCta aattggittitt CCt. Ctct CC aaaataagtg aaagtagat.ca gtttalaaggC 841 tCtttgcaa.g tatttgtgct tagagtacaa agagtaaaat attt tatgtt Cattatttaa. 901 aagttaaaat attcaattaa. tCaaaatatt ttattattaa atata attic CCaataaaaa 96. atttittataa aCCatatoat aattittggta Cattitatgag aaattgttgaC tgtttgaaaa. 102. ttgattgitat CCCalaa Catt gtggtaCCaa tgaCtattitc agt Citaa CCt aCatgggCtC 108 tgattaaata galagataCat totttgttc attaacaa at aatgtcateg aattita Cagt 41 atgtttatat Catgtcaaag atatgcatgt. aaagatgctC tggttaggta aact Cttaaa 1201. atttcattt C tgCttctgga aaattaggat. aaatggat Ct tCtta gaatt atttatagaa 26 aatgatatala aattattaac tattagggac agattaatta talagtotgtc aagCaggcaa. 32 aggtoagaaa aaaatcggtg tgttactCaa attatC gatg gttgtaaagg ttittaaag Ca 1381 gggCtgccCa Cataga CaCt tCaaatataa tgataaagtag acaaa CCttt tggaaattat 1441 aaattittaaC ttaCtggitta CcatCtttitc tagtaaaact gaagatggat. tCitctagtaa 15C1 tatttgtaala ttaCatgttc attttgttitt ttitcCC Cag FIG. 3 U.S. Patent Mar. 2, 1999 Sheet 4 of 13 5,876,942 OCUS BOWAVE 1684 bp dis-DNA 28-OCT-1995 ACESSIONDEFINITION L48508Bos taurus X chromosome anielRelogenin o (AMLX) gene, complete intron 5. Eps settiOS taurus (strain(Strain HolHolstein) ) adult blb ORGANISM Bos taurus OOd DNA Eukaryota; Animalia; Chordata; Vertebrata; Marmalia; Theria; Elia; Artiodactyla; Ruminantia; Pecora; Bovidae; Bovinae; OW REEE (bases to 684) TITLEA9RS ChengCheng, paperW.T.K., Chen, C.-H., Hu, C.-L., Wang, C.-H. and Choo, K.-B. JOURNAL Unpublished (1995) STANDARD full staff review REFERENCE 2 (sites) AUTHORS Cheng, W.T.K. TITLE No Title) JOURNAL Unpublished (1995) STANDARD full staff review FEATURES Location/Qualifiers introl ... 1684 /number=5 /map=" chromosose X" A gene="AMLX" evidence=EXPERIMENTAL Y /citations (2) miSC difference ... 1684 /note="65. 9 Sequence similarity with bovine Y chromosome amelogenin gene (bAMLY gene) intron 5 feitation= (2)A. SOCe ... 1684 /organisa="Bos taurus" /strain="Holstein." ?cell type="leukocyte" /dev stage="adult" /germline /acronuclear(sequenced-moi"DNA /tissue-type="blood" BASE COUNT 542 a. 265 C 327 g 550 t. ORIGIN gtgagtata C CttgaggtCa Cta CaaCaCt. tatgaaaatg gtC gag.cgaa aatggCCCCC 6. Ccagagattg aaaatctotC acagte Caag gCCta Cagtt toagtag Cita Cagcta Cagc 21 tCagtgattC agttagcCCa agtgttgttgct gtaagttitat attataaaga gttCatctat 181 atggCttaga attaaCaCaa tagct ttitat ggtgga Cagg gaggCCtggC gtgCtgCgat 24 tCatggggtC gtaaagagtC ggaCatgact gagctactga tCtgatCtga tCtgaaattic 30 tataattact ttgaattCct aCCagaatat Citat tattga aacct gttaa. ttitta CagaC 36. agtactataa gggaaCatta gttct Ctta tCtttcaaag gtttgaCCag Caataatagg 42 Ctagaattga accggagatt tCttittaaaa. gggtaattit tgC Cagt gag gggatgcaga 48 tatttgaa.ca ggtaggtggg aggtCtCttt aaaaac Citaa aggaaaatta Cttgtttaaa 541 gtggttt toa at CtaaCttg gagga agcta aaCtaCaCag attgCtagat tCCttt toag 60. tgctgggatt Ctgtagatta a gCttittagC aataagtCtt Catagitaaag gaCaaaatat 66 CCttt Catta CagaaagctC titt CCCaCtC tt CCaagtga gtgggaaata gt. Caataag. 721 gaCCCttatC Catatatata ala CCCtCact tgttgttgtata agagattatg taattCtact 781 gtatgcatgt Cttcaaatgt. aattCCaag C tataaatgtt attaatgatt. ttCtaCtgag 84l aaggtgaggg gtggggtaga aatga CCtgg atggggagcC gggataCCtg agitt CCagtt 30 ataactCtgC actCaaaaag gagatgCtgt. ttaa CaagtC act tcaacta tatgggtgtt 96. taCCCgittaa aatgagagga. agtaaCtaaa tCaaataaCt tt CaagcCtt tgtCtaagaa 102. gaaataCCat agtttitcCaa taaaaattat atgttatata aggCttcaaa. attatgggaa 08 tatttgttgga gala CaCaggC at atttgttgg aaaatatgga gCatatttga aaaatgattg 14 tat CCCaaat gctCtggtgC Cta Cagttga ttatt Ctagt Cta aggta Ca ggttaCEgt. Ct. 201 ttgtatagaa aatatgttta tCtCgCCaaa at gCattitat Ctct t tatta ataattagtg 26 titatgaactt. tataaat Ctt. gCC a gag Ca ttcaaggaaa ggtgattittg ggtaCtagt 1321 ttgaactCtt taaaattcCC at Ctaaaatt a gaggadat aggatagc3 tCttagag, 381 gtCttaagaa aataaagtgt. tgagtttaag aaat, gaCCat gCagaaagta Ct. CaCCCCaa l44l taCagcaatt gttgtttitat tatt Caataa ttgttt taag tatt Citgaag gCaggggcCt. 150 CCatCctgaC acct Catatc taatgacitat aga Cala Ca.3 agaaagtttg tgaattatag 156 agttcaactt CaatggCtat aatattgttt tggCaaaatt gaaCCCatat ttgttaCtat 162 aaatggtact Cactaagaat atttgta aat tgttittaCtt gCttCttittg Caattittt tt 1681 -c.ca.g U.S. Patent Mar. 2, 1999 Sheet 5 of 13 5,876,942 8 U.S. Patent Mar. 2, 1999 Sheet 6 of 13 5,876,942 ts U.S. Patent Mar. 2, 1999 Sheet 7 of 13 5,876,942 U.S. Patent Mar. 2, 1999 Sheet 9 of 13 5,876,942 89|-! U.S. Patent Mar. 2, 1999 Sheet 10 Of 13 5,876,942 occatacTaggac-TacACACAGAAAATCGCGAccAAAATCGCescarawcreecreccacci 99 it AAGCCAAcacACAAcAAAAAAACAccaricca-Acware AATecrecrecciac 9. coacaccacaccarocracAGGGTCCTGVWTTAAAAvalorexicanarcircucca 9 Y calc-s-s-ArcAAAccAGAAAAccessal