IP Library › Granted Patent US 12,258,630
Granted Patent B2
US 12,258,630 · App. 17/178,806 · Granted Mar 25, 2025

Methods and systems for assessing and/or quantifying sperm cell subpopulations bearing a specific genetic signature

Inventors: Elon Roti-Roti (Windsor, WI); Nicole Cray (Madison, WI); Matthias Wagner (Cambridge, MA); Michael Reid Botts (DeForest, WI)
Assignee: ABS Global, Inc.
C12Q1/6879C12Q1/6841C12Q2600/124C12Q2600/16C12Q2600/166
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Quick Facts
Patent No.
US 12,258,630
App. No.
17/178,806
Granted
Mar 25, 2025
Kind
B2
Abstract

Technologies for assessing, quantifying and isolating sperm cell populations and/or subpopulations having specific genetic signatures are provided, as well as methods and systems to assess the efficacy of chromosomal differentiation processes. Compositions for identification and differentiation of X-chromosomes and Y-chromosomes in DNA are also provided.

Claims (27)

1. A system for assessing the sex-skew in a population of cells, comprising:

a first oligonucleotide agent that selectively binds to an X-chromosome nucleic acid sequence comprising at least one sequence selected from the group consisting of SEQ ID NO: 4-6, 28-35, 222, and 845-847; and

a second oligonucleotide agent that selectively binds to a Y-chromosome nucleic acid sequence comprising at least one sequence selected from the group consisting of SEQ ID: 7-9, 36-55, 767, 850-861, and 864-875.

2. The system of claim 1 , further comprising a third oligonucleotide agent that selectively binds to an autosomal chromosome nucleic acid sequence.

3. The system of claim 1 , wherein:

said first nucleotide agent comprises oligonucleotide primer sequences SEQ ID NOs: 4 and 5, and oligonucleotide probe sequence SEQ ID NO: 6; and

said second nucleotide agent comprises oligonucleotide primer sequences SEQ ID NOs: 7 and 8, and oligonucleotide probe sequence SEQ ID NO: 9.

4. The system of claim 3 , further comprising a third nucleotide agent comprising oligonucleotide primer sequences SEQ ID NOs: 1 and 2, and oligonucleotide probe sequence SEQ ID NO: 3.

5. The system of claim 1 , further comprising reagents and buffers for removing non-viable sperm cells.

6. The system of claim 1 , further comprising reagents and buffers for isolating DNA from a sample of sperm cells.

7. The system of claim 1 , wherein the oligonucleotide agent is contained in an oligo-chip.

8. The system of claim 1 , wherein the oligonucleotide agent comprises a detectable label.

9. The system of claim 8 , wherein the detectable label is selected from the group consisting of a fluorochrome and a radioactive isotope.

10. The system of claim 8 , wherein the detectable label is selected from the group consisting of fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 32 P, 35 S, and 3 H.

11. The system of claim 1 , wherein the system further comprises a droplet generator and a droplet reader.

12. The system of claim 1 , wherein the system further comprises a cytometer.

13. The system of claim 1 , further comprising dNTPs and polymerase enzymes.

14. A method of assessing sex-skew in a population of sperm cells, the method comprising steps of:

detecting binding of a set of oligonucleotide agents to sperm DNA obtained from a sample of sperm cells, wherein the set includes:

a first oligonucleotide agent that selectively binds to an X-chromosome nucleic acid sequence comprising at least one sequence selected from the group consisting of SEQ ID NO: 4-6, 28-35, 222, and 845-847; and

a second oligonucleotide agent that selectively binds to a Y-chromosome nucleic acid sequence comprising at least one sequence selected from the group consisting of SEQ ID: 7-9, 36-55, 767, 850-861, and 864-875.

15. The method of claim 14 , further comprising isolating sperm DNA from a population of sperm cells from a semen sample.

16. The method of claim 15 , further comprising removing laser ablated sperm cells from said semen sample of sperm cells prior to said isolating sperm DNA.

17. The method of claim 1 , wherein said population of cells comprise an X-skewed sperm population.

18. The method of claim 14 , wherein said step of detecting comprises amplifying at least one of an X-chromosome target site and a Y-chromosome target site.

19. The method of claim 15 , wherein said semen sample is from Bos taurus, Bos indicus, Bos bubalis , or hybrids thereof.

20. The method of claim 14 , wherein said detecting comprises performing multiplex ddPCR.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 20, 2021
From: GENUS PLC
To: ABS GLOBAL, INC.
Reel/Frame 055975/0885 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 18, 2021
From: ROTI-ROTI, ELON; CRAY, NICOLE; WAGNER, MATTHIAS; BOTTS, MICHAEL REID
To: GENUS PLC
Reel/Frame 055321/0865 →
Continuity (3)
Continuation 16121077 · Sep 4, 2018
Provisional Application 62553771 · Sep 1, 2017
Related Publication 20210189488A1 · Jun 24, 2021
References Cited (125)
US 3687803A · Grayson et al. · 1972 [cited by applicant]
US 3894529A · Shrimpton et al. · 1975 [cited by applicant]
US 4009260A · Ericsson · 1977 [cited by applicant]
US 4067965A · Bhattacharya · 1978 [cited by applicant]
US 4083957A · Lang · 1978 [cited by applicant]
US 4085205A · Hancock · 1978 [cited by applicant]
US 4092229A · Bhattacharya · 1978 [cited by applicant]
US 4155831A · Bhattacharya · 1979 [cited by applicant]
US 4191749A · Bryant · 1980 [cited by applicant]
US 4225405A · Lawson · 1980 [cited by applicant]
US 4276139A · Lawson · 1981 [cited by applicant]
US 4339434A · Ericsson · 1982 [cited by applicant]
US 4448767A · Bryant · 1984 [cited by applicant]
US 4511661A · Goldberg · 1985 [cited by applicant]
US RE32350E · Bhattacharya et al. · 1987 [cited by applicant]
US 4680258A · Hammerling et al. · 1987 [cited by applicant]
US 4683195A · Mullis et al. · 1987 [cited by applicant]
US 4683202A · Mullis · 1987 [cited by applicant]
US 4698142A · Muroi et al. · 1987 [cited by applicant]
US 4749458A · Muroi et al. · 1988 [cited by applicant]
US 4988167A · Fergason · 1991 [cited by applicant]
US 4999283A · Zavos et al. · 1991 [cited by applicant]
US 5021244A · Spaulding · 1991 [cited by applicant]
US 5135759A · Johnson · 1992 [cited by applicant]
US 5346990A · Spaulding · 1994 [cited by applicant]
US 5439362A · Spaulding · 1995 [cited by applicant]
US 5459038A · Reed · 1995 [cited by applicant]
US 5514537A · Chandler · 1996 [cited by applicant]
US 5596089A · Silversides · 1997 [cited by applicant]
US 5660997A · Spaulding · 1997 [cited by applicant]
US 6149867A · Seidel · 2000 [cited by applicant]
US 8858943B2 · Burbidge · 2014 [cited by applicant]
US 9588100B2 · Appleyard et al. · 2017 [cited by applicant]
US 9683922B2 · Wagner et al. · 2017 [cited by applicant]
US 10208350B2 · Beim · 2019 [cited by applicant]
US 10961577B2 · Roti-Roti · 2021 [cited by examiner]
US 20040049801A1 · Seidel · 2004 [cited by applicant]
US 20040053243A1 · Evans · 2004 [cited by applicant]
US 20050130115A1 · Funk · 2005 [cited by applicant]
US 20090087847A1 · Lo · 2009 [cited by applicant]
US 20090227606A1 · DeLeo · 2009 [cited by applicant]
US 20100029498A1 · Gnirke · 2010 [cited by examiner]
US 20100260670A1 · Zeiger · 2010 [cited by applicant]
US 20140182005A1 · Oksenberg · 2014 [cited by applicant]
US 20140275219A1 · Cao · 2014 [cited by applicant]
US 20160223442A1 · Guldberg · 2016 [cited by examiner]
US 20170204370A1 · Morjal et al. · 2017 [cited by applicant]
US 20170226594A1 · Altayari · 2017 [cited by examiner]
US 20190025212A1 · Evans · 2019 [cited by applicant]
EP 3323828 · 2018 [cited by applicant]
KR 20170008181A · 2017 [cited by applicant]
WO WO9511995 · 1995 [cited by applicant]
WO WO9933956 · 1999 [cited by applicant]
WO WO0006193 · 2000 [cited by applicant]
Aasen et al., Amplification of ZFY and ZFX genes for sex determination in humans, cattle, sheep and goats. Biotechnology 8:1279. (1990). [cited by applicant]
Ali et al., Enrich. of Bovine X-and Y-Chrom-Bearing Sperm with Monoclonal H-Y Antibody-Fluorescence-Activated Cell Sorter. Archives of Andrology 24:235 (1990). [cited by applicant]
Bilal et al., Buffalo : Black gold of Pakistan. Livestock Research for rural development 18(9) (2006). [cited by applicant]
Canavez et al., Genome sequence and assembly of Bos indicus. J. of Heredity 103(3) : 342 (2012). [cited by applicant]
Enciso et al., The ability of sperm selection techniques to remove single—or double-strand DNA damage. Asian J. of Andrology 13: 764-768 (2011). [cited by applicant]
Fernandez et al., The Effect of Low-Level Laser Irradiation on Sperm Motility, and Integrity of the Plasma Membrane and Acrosome in Cryopreserved Bovine Sperm. Plos One | DOI: 10.1371 Journal.pone.0121487 (Mar. 2015) 11… [cited by applicant]
Gravitt et al., Reproducibility of HPV 16 and HPV 18 viral load quantitation using TaqMan real-time PCR assays. J. of Virologicall Methods 112:233-33 (2003). [cited by applicant]
Johnson et al., Sex Preselection in Rabbits : Live Births from X and Y Sperm separated by DNA and cell sorting. Biology of Reproduction 41:199-203 (1989). [cited by applicant]
Kierstein et al., Analysis of mitochondrial D-loop region casts new light on domestic water buffalo (Bubalus bubalis) phylogeny. Molecular Phylogenetics and Evolution 30:308-324 (2004). [cited by applicant]
Liu et al. Bos taurus genome assembly. BMC Genomics 10:180 (2009). [cited by applicant]
Lun et al., Microfluidics Digital PCR Reveals a Higher than Expected Fraction of Fetal DNA in Maternal Plasma. Clinical Chemistry 54(10): 1664 (2008). [cited by applicant]
Peipo et al., Birth of Correctly Genotyped Calves After Multiplex Marker Detection From Bovine Embryo Microblade Biopsies. Molecular Reproduction and Development 74: 1373-1378 (2007). [cited by applicant]
Pruitt et al., NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Research 35: Database Issue D61-D65 (2007). [cited by applicant]
Rychlik et al., A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Research 17(21) : 8543 (1989). [cited by applicant]
Schneider-Gadicke et al., ZFX has a gene structure similar to ZFY, the putative human sex determinant, and escapes X inactivation. Cell 57 : 1247 (1989). [cited by applicant]
Thellin et al. Housekeeping genes as internal standards: use and limits. J. of Biotechnology 75:291 (1999). [cited by applicant]
Vitra et al., Sex determination of bovine embryo blastomeres by fluorogenic probes. Theriogenology 57: 2229-2236 (2002). [cited by applicant]
Welch et al., Flow cytometic sperm sorting and PCR confirm separation of X- and Y-chromosome bearing bovine sperm. Animal Biotechnology 6(2) : 131-139 (2009). [cited by applicant]
Kirkpatrick et al., Sensitive sex determination assay applicable to bovine embryos derived from IVM and Ivf. J. of Reproduction and Fertility 98 : 335 (1993). [cited by applicant]
Zimin et al., A whole-genome assembly of the domestic cow, Bos taurus. Genome Biology 10:R42 (2009). [cited by applicant]
Floren et al., Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR). Food Chemistry 173:1054-1058 (2015). [cited by applicant]
Kirkness et al., The Dog Genome : Survey Sequencing and comparative Analysis. Science 301 : 1898 (2003). [cited by applicant]
Nielsen et al., A Scan for Positively Selected Genes in the Genomes of Humans and Chimpanzees. PLoS Biology 3 (6) :e170 (2005). [cited by applicant]
Smith et al., Sequence Evaluation of Four Pooled-Tissue Normalized Bovine cDNA Libraries and Construction of a Gene Index for Cattle. Genome Research 11: 626 (2001). [cited by applicant]
Yu et al., Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients. Molecular Medicine Reports 16:1157… [cited by applicant]
Helton et al., Selection and use of SNP markers for animal identification and paternity analysis in U.S. beef cattle. Mammalian Genome 13:272-281 (2002). [cited by applicant]
Smith et al., Sequence Evaluation of Four Pooled-Tissue Normalized Bovine cDNA Libraries and Construction of a Gene Index for Cattle. Genome Research 11: 626-630 (2001). [cited by applicant]
International Search Report and Written Opinion of the International Searching Authority dated Dec. 10, 2018; International Appln. No. PCT/IB2018/056710, Filed Sept. 3, 2018, Applicant: Genus plc. [cited by applicant]
U.S. Appl. No. 60/211,093, filed Jun. 12, 2000, Whittier et al. [cited by applicant]
U.S. Appl. No. 60/224,050, filed Aug. 9, 2000, Whittier et al. [cited by applicant]
U.S. Appl. No. 60/253,785, filed Nov. 29, 2000, Seidel et al. [cited by applicant]
Borchersen et al., “Danish A.I. field data with sexed semen,” [cited by applicant]
Cerchiaro et al., “A field Study on fetility and purity of sex-sorted cattle sperm.,” [cited by applicant]
Chen et al., “Single nucleotide poiymorphism genotyping: biochemistry, protocol, cost and throughput” [cited by applicant]
De Risi et al., “Use of a cDNA microarry to analyse gene expression patterns in human cancer.” [cited by applicant]
Deiahunty et al., “Testing the Feasibility of DNA Typing for Human Identification by PCR and an Oligonucleotide Ligation Assay,” [cited by applicant]
Egholm, et al., “PNA hybridizes to complementary oligonucleotides obeyign the Watson-Crick hydrogen-bonding rules,” [cited by applicant]
Faust et al., “1133 Effects for fertility of processing steps of a new technology platform for producing sexed sperm” [cited by applicant]
Habermann et al., “Validation of sperm sexing in the cattle (Bos taurus) by dual colour fluorescence in situ hybridization,” [cited by applicant]
Hacia et al., “Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour fluoresence analysis,” [cited by applicant]
Healy et al., “Artificial insemination field data on the use of sexed and conventional semen in nulliparous Holstein heifers,” [cited by applicant]
Hindson et al., “High-throughput droplet digital PCR system fo absolute quantitation of DNA copy number,” [cited by applicant]
International Search Report mail on Dec. 10, 2018, in Internationai Patent Application No. PCT/IB2018/056710. [cited by applicant]
Joerg et al., “Validating bovine sexed semen samples using quantitative PCR,” [cited by applicant]
Johnson et al., “Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum eficiency” [cited by applicant]
Johnson, “Sex preselection by flow cytometric separation of X and Y chromosome-bearing sperm based on DNA difference: a review,” [cited by applicant]
Kawarasaki et al., “Verification of flow cytometorically-sorted X- and Y- bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the floresence in situ hybridization (FISH) technique,” [cited by applicant]
Khalajzadeh et al., “Effect of widespread and limited use of sexed semen on genetic progress and reproductive performance of dairy cows,” [cited by applicant]
Khamlor et al., “Determination of Sperm Sex Ratio in Bovine Semen Using Multiplex Real-time Polymerase Chain Reaction,” [cited by applicant]
Kwok et al., “Detection of single nucleotide polymorphisms,” [cited by applicant]
Kwok, “Methods for genotyping single nucleotide polymorphisms,” [cited by applicant]
Lalande et al., “Quantitative studies on the precursors of cytotoxic lymphocytes, VI. Second signal requirements of specifically activated precusors isolate 12 h after stimulation,” [cited by applicant]
Lalande et al., “Fluorescence flow analysis of lymphocyte activation using Hoechst 33342 dye,” [cited by applicant]
Lockhart et al., “Expression monitoring by hybridization to high-density oligonucleotide arrays,” [cited by applicant]
Loken, “Separation of viable T and B lymphocytes using a cytochemical stain, Hoechst 33342,” [cited by applicant]
Loken, “Simultaneous Quantitation of Hoechst 33342 and Immunofluorescence on Viable Cells using a Fluorescence Activated Cell Sorter,” [cited by applicant]
Mateizel et al., “FISH analysis of chromosome X, Y and 18 abnormalities in testicular sperm from azoospermic patients,” [cited by applicant]
Moruzzi, “Selecting a mammalian species for the separation of X- and Y-chromosome-bearing spermatozoa,” [cited by applicant]
Norman et al., “Use of sexed semen and its effect on conception rate, calf sex, dystocia, and stillbirth of Holsteins in the United States,” [cited by applicant]
Ørum et al., “Single base pair mutation analysis by PNA directed PCR clamping,” [cited by applicant]
Parati et al., “Sex ratio determination in bovine semen: a new approach by quantitative real time PCR” [cited by applicant]
Puglisi et al., “In vitro fertilisation with frozen-thawed bovine sperm sexed by flow cytometry and validated for acuracy by real-time PCR” [cited by applicant]
Ramsay, “DNA chips: State-of-the art,” [cited by applicant]
Rens et al., “An X-Y paint set and sperm FISH protocol that can be used for validation of cattle sperm separation procedures,” [cited by applicant]
Riley et al., “A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones,” [cited by applicant]
Saiki et al., “Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia,” [cited by applicant]
Seidel, “Update on sexed semen technology in cattle,” [cited by applicant]
Shi, “Technologies for individual genotyping: detection of genetic polymorphisms in drug targets and disease genes,” [cited by applicant]
Smith et al., “Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology,” [cited by applicant]
Uhlmann et al., “Antisense oligonucleotides: a new therapeutic principle,” [cited by applicant]
Van Munster et al., “Difference in volume of X- and Y-chromosome-bearing bovine sperm heads matches difference in DNA content,” [cited by applicant]