Y-chromosome specific polynucleotide probes for prenatal sexing

• US 5,663,048 A
• Filed: 10/27/1994
• Issued: 09/02/1997
• Est. Priority Date: 10/04/1990
• Status: Expired due to Fees

• Current Assignee: University of Calgary
• Original Assignee: University of Calgary
• Inventors: Winkfein, Robert J., Dixon, Gordon H.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CAMPBELL, EGGERTON A
• Application Number:
  US08/330,537
• Time in Patent Office: 1,041 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78, 536/24.3, 536/24.31
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/8771 Bovine embryos
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Y-chromosome specific hybridization probes for prenatal sexing are provided capable of hybridizing only to Y-chromosome specific DNA sequences of bovine and other ruminants and are suitable for sexing embryos at or before the time of embryo transfer with essentially 100% accuracy.

Claims

1. A labeled or unlabeled Y-chromosome specific nucleic acid isolate which comprises the same sequence as that of either strand of an isolate selected from the group consisting of:

(1) one or both strands of the PstI fragment SEQ ID NO;

1;

BtY1, and

(2) one of both strands of the SacI fragment SEQ ID NO;

3;

BtY2.

2. The nucleic acid according to claim 1 which is radioactively labeled with ³ H, ³⁵ S, ³² P, or ¹²⁵ I.

3. The nucleic acid according to claim 1 which is non-radioactively labeled with biotin or bromodeoxyuridine.

4. The nucleic acid according to claim 1 wherein said nucleic acid is DNA.

5. The nucleic acid according to claim 1 wherein the nucleic acid is RNA.

6. The nucleic acid according to claim 4 which is radioactively labeled with ³ H, ³⁵ S, ³² P, or ¹²⁵ I.

7. The nucleic acid according to claim 4 which is non-radioactively labeled with biotin or bromodeoxyuridine.

8. The nucleic acid according to claim 5 which is radioactively labeled with ³ H, ³⁵ S, ³² P, or ¹²⁵ I.

9. The nucleic acid according to claim 5 which is non-radioactively labeled with biotin or bromodeoxyuridine.

10. The nucleic acid according to claim 4 wherein said DNA comprises the same sequence as that of either strand of a DNA selected from the group consisting of closed circular SEQ ID NO:

1, BtY1 and linearized SEQ ID NO;

1, BtY1.

11. The nucleic acid according to claim 10 which is radioactively labeled with ³ H, ³⁵ S, ³² P, or ¹²⁵ I.

12. The nucleic acid according to claim 10 which is non-radioactively labeled with biotin or bromodeoxyuridine.

13. The nucleic acid according to claim 4 wherein said DNA comprises the same sequence as that of either strand of a DNA selected from the group consisting of closed circular SEQ ID NO:

3, BtY2 and linearized SEQ ID NO;

3, BtY2.

14. The nucleic acid according to claim 13 which is radioactively labeled with ³ H, ³⁵ S, ³² P, or ¹²⁵ I.

15. The nucleic acid according to claim 13 which is non-radioactively labeled with biotin or bromodeoxyuridine.

16. A replicable vector comprising the nucleic acid isolate according to claim 1.

18. A method for the determination of the presence or absence of a Y-chromosome in a ruminant, said method comprising:

isolating DNA from a tissue or cell sample of a ruminant,

immobilizing said DNA onto a support matrix,

hybridizing the immobilized DNA with a nucleic acid isolate of claim 1 under conditions allowing the nucleic acid isolate to bind to complementary sequences,

washing unbound nucleic acid isolate from the support matrix, and

detecting nucleic acid isolate binding to DNA immobilized on the support matrix.

19. A method for determining the presence or absence of a Y-chromosome in fixed cells or chromosomes in the interphase or metaphase stages of nuclear division of a ruminant said method comprising:

hybridizing said fixed cells or interphase or metaphase chromosomes of a ruminant with the nucleic acid isolate of claim 1 under conditions allowing the nucleic acid isolate to bind to complementary sequences,

washing away unbound nucleic acid isolates, and

detecting binding of the nucleic acid isolate to the complementary sequences in the fixed cells or interphase or

detecting binding of the nucleic acid isolate to the complementary sequences in the fixed cells or interphase or metaphase chromosomes.

20. The method for determining the presence or absence of a Y-chromosome in a tissue or cell sample of a ruminant comprising:

isolating DNA from a tissue or cell sample or a ruminant and denaturing the isolated DNA,

annealing the denatured DNA with a synthetic polynucleotide comprising 12 or more nucleotides from the nucleic acid isolate of claim 1 under conditions allowing the synthetic polynucleotide to bind to a target DNA,

amplifying the target DNA,

immobilizing the amplified target DNA onto a support matrix,

hybridizing the immobilized amplified target DNA with the nucleic acid isolate of claim 1 under conditions allowing the nucleic acid isolate to bind to complementary sequences,

washing unbound nucleic acid isolate from the support matrix, and

detecting binding of the nucleic acid isolate.

21. A method for determining the presence or absence of Y-chromosomes in a tissue or cell sample of a ruminant comprising:

isolating DNA from a tissue or cell sample of a ruminant and denaturing the isolated DNA,

annealing the denatured DNA with a synthetic polynucleotide comprising 12 or more nucleotides from the nucleic acid isolate of claim 1 under conditions allowing the synthetic polynucleotide to bind to a target DNA,

incubating labeled nucleotide precursors with a DNA polymerase,

amplifying the target DNA,

hybridizing an amplified target DNA with a nucleic acid isolate of claim 1 under conditions allowing binding to complementary sequences,

fractionating the sample by electrophoresis in a gel matrix, and

detecting labeled nucleic acid isolates.

22. A method according to claim 18 wherein the nucleic acid isolate is labeled with a detectable marker selected from ³ H, ³⁵ S, ³² P, ¹²⁵ I, biotin or bromodeoxyuridine.

23. A method according to claim 19 wherein the nucleic acid isolate is labeled with a detectable marker selected from ³ H, ³⁵ S, ³² P, ¹²⁵ I, biotin or bromodeoxyuridine.

24. A method according to claim 20 wherein the nucleic acid isolate is labeled with a detectable marker selected from ³ H, ³⁵ S, ³² P, ¹²⁵ I, biotin or bromodeoxyuridine.

25. A method according to claim 21 wherein the nucleic acid isolate is labeled with a detectable marker selected from ³ H, ³⁵ S, ³² P, ¹²⁵ I, biotin or bromodeoxyuridine.

26. A kit for detecting the presence or absence of Y-chromosome specific sequences in a tissue or cell sample of a ruminant comprising the nucleic acid isolate according to claim 1 and a buffer.

27. The method of claim 18 wherein the ruminant is selected from the group consisting of bovine, ovine and caprine.

28. The method of claim 19 wherein the ruminant is selected from the group consisting of bovine, ovine and caprine.

29. The method of claim 20 wherein the ruminant is selected from the group consisting of bovine, ovine and caprine.

30. The method of claim 21 wherein the ruminant is selected from the group consisting of bovine, ovine and caprine.

17. A replicable vector comprising a nucleic acid isolate encoding SEQ ID NO:

3, BtY2.