Nucleic acid probes for prenatal sexing

• US 4,960,690 A
• Filed: 06/05/1986
• Issued: 10/02/1990
• Est. Priority Date: 05/31/1985
• Status: Expired due to Fees

• Current Assignee: Merck & Co., Inc.
• Original Assignee: Salk Institute Biotechnology Industrial Associates, Inc.
• Inventors: Ellis, Steven B., Harpold, Michael M.
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): Wagner, Richard
• Application Number:
  US07/871,111
• Time in Patent Office: 1,580 Days
• Field of Search: 435/6, 435/172.3, 436/501, 436/814, 536/27, 935/3, 935/9, 935/63, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: Y10S 436/814 Pregnancy

Abstract

Nucleic acid hybridizaiton probes are provided which have sequences complementary to sequences of segments in bovine male-specific DNA and are suitable for sexing bovine embryos at the time of embryo transfer with nearly 100% accuracy.

Claims

1. A labeled or unlabeled single-stranded nucleic acid which comprises:

(a) a segment which has substantially the same sequence as that of either strand of the 6.2 kbp EcoRI fragment of the genome of lambda-ES6.0;

or

(b) any segment, more than 20 bp in length, of the segment of subparagraph (a) of this Claim;

provided that said single-stranded nucleic acid hybridizes to a significantly greater extent with total DNA of males of a breed of a species of genus Bos than with total DNA of females of the breed.

2. A nucleic acid according to claim 1 which, under stringent conditions, hybridizes to a significantly greater extent with total DNA of males of a breed selected from the group consisting of Hereford and Holstein than with total DNA of females of the breed.

3. A nucleic acid according to claim 2 which is DNA.

4. A DNA according to claim 3 which has substantially the same sequence as that of either strand of a DNA selected from the group consisting of the genome of lambda-ES6.0, the 6.2 kbp EcoRI fragment of the genome of lambda-ES6.0, the 4.0 kbp EcoRI fragment of the genome of lambda-ES6.0, and the 2.2 kbp EcoRI fragment of the genome of lambda-ES6.0.

5. A DNA according to claim 4 which is either strand of a DNA selected from the group consisting of the genome of lambda-ES6.0, the 6.2 kbp EcoRI fragment of the genome of lambda-ES6.0, the 4.0 kbp EcoRI fragment of the genome of lambda-ES6.0, and the 2.2 kbp ECoRI fragment of the genome of lambda-ES6.0.

6. A nucleic acid according to claim 1 which is radioactively labeled with ³ H or ³² P.

7. A nucleic acid according to claim 1 which is non-radioactively labeled with biotin.

8. A nucleic acid according to claim 2 which is radioactively labeled with ³ H or ³² P.

9. A nucleic acid according to claim 2 which is non-radioactively labeled with biotin.

10. A DNA according to claim 3 which is radioactively labeled with ³ H or ³² P.

11. A DNA according to claim 3 which is non-radioactively labeled with biotin.

12. A DNA according to claim 4 which is radioactively labeled with ³ H or ³² P.

13. A DNA according to claim 4 which is non-radioactively labeled with biotin.

14. A DNA according to claim 5 which is radioactively labeled with ³ H or ³² P.

15. A DNA according to claim 5 which is non-radioactively labeled with biotin.

16. A labeled or unlabeled single-stranded DNA which is more than 20 bp in length, which under stringent conditions hybridizes to a significantly greater extent with total DNA of males of a breed of genus Bos selected from the group consisting of Hereford and Holstein than with total DNA of females of the breed, and which is made by the process of denaturing DNA made by nick-translation of a DNA selected from the group consisting of plasmid pES5(2), plasmid pES8 and the genome of lambda-ES6.0.

17. A DNA according to claim 16 which is radioactively labeled with ³ H or ³² P.

18. A DNA according to claim 16 which is non-radioactively labeled with biotin.

19. E. coli LE392 (pES5(2) or E. coli LE392 (pES8).

20. Phage lambda-ES6.0.

21. A double-stranded DNA which has substantially the same sequence as a DNA selected from the group consisting of the genome of lambda-ES6.0, the 6.2 kbp EcoRI fragment of the genome of lambda-ES6.0, the 4.0 kbp EcoRI fragment of the genome of lambda-ES6.0, and the 2.2 kbp EcoRI fragment of the genome of lambda-ES6.0.

22. A DNA according to claim 21 which is a DNA selected from the group consisting of the genome of lambda-ES6.0, the 6.2 kbp EcoRI fragment of the genome of lambda-ES6.0, the 4.0 kbp EcoRI fragment of the genome of lambda-ES6.0, and the 2.2 kbp EcoRI fragment of the genome of lambda-ES6.0.

23. A nucleic acid according to claim 21 which is radioactively labeled with ³ H or ³² P.

24. A nucleic acid according to claim 21 which is non-radioactively labeled with biotin.

25. A nucleic acid according to claim 22 which is radioactively labeled with ³ H or ³² P.

26. A nucleic acid according to claim 22 which is non-radioactively labeled with biotin.

27. A method of sexing an embryo or fetus of a species of a genus Bos which comprises:

(i) contacting the DNA of cells of said embryo or fetus under hybridization conditions with one or more hybridization probes, each of which is a detectably labeled, single-stranded nucleic acid which comprises;

(a) a segment which has substantially the same sequence as that of either strand of a fragment selected from the group consisting of;

(1) the smaller PstI fragment of plasmid pES5(2),

(2) the smaller PstI fragment of plasmid pES8, and

(3) the 6.2 kbp EcoRI fragment of the genome of lambda-ES6.0;

or

(b) any segment, more than 20 bp in length, of the segment of subparagraph (i)(a) of this claim;

provided that at least one of said single-stranded nucleic acids, under the hybridization conditions, hybridizes to a significantly greater extent with total DNA of males of a breed of a species of the genus Bos than with total DNA of females of the breed; and

(ii) ascertaining whether significant hybridization occurs between the DNA of the cells of the embryo or fetus and the hybridization probe or probes, wherein significantly greater hybridization is an indication of the presence of a male embryo or fetus.

28. A method according to claim 27 wherein each of the hybridization probes is a nucleic acid which is radioactively labeled with ³² P or ³ H and which hybridizes under stringent conditions to a significantly greater extent with total DNA of males of a breed selected from the group consisting of Hereford and Holstein than with total DNA of females of the breed.

29. A method according to claim 28 wherein each of the hybridization probes is a DNA and is obtained by denaturing DNA made by nick-translation of a DNA with a sequence which is substantially the same as that of a DNA selected from the group consisting of closed circular pES5(2), closed circular pES8, linearized pES5(2), linearized pES8, the smaller PstI fragment of pES5(2), the smaller PstI fragment of pES8, the genome of lambda-ES6.0, the 6.2 kbp EcoRI fragment of the genome of lambda-ES6.0, the 4.0 kbp EcoRI fragment of the genome of lambda-ES6.0, and the 2.2 kbp EcoRI fragment of the genome of lambda-ES6.0.

30. A method according to claim 29 wherein each of the hybridization probes is obtained by denaturing DNA made by nick-translation, with one or more of the ³² P-labeled deoxynucleoside triphosphates, of a DNA selected from the group consisting of plasmid pES5(2), plasmid pES8 and the genome of lambda-ES6.0.

31. A method according to claim 30 wherein the DNA probed is derived from at least 4 embryonic cells, the hybridization probes are a mixture of fragments obtained by nick-translation of pES5(2), pES8, and the genome of lambda-ES6.0 with all four deoxynucleoside triphosphates labeled with ³² P, and the radioactively of probes derived from each of pES5(2), pES8 and the genome of lambda-ES6.0 is at least one fifth and no more than one half of the radioactivity of the mixture.

32. A method according to claim 31 wherein the DNA probed is derived from at least 10 embryonic cells.

33. A method according to claim 27 wherein one or more of the hybridization probes is a nucleic acid which is non-radioactively labeled with biotin and wherein each of the probes is a nucleic acid which hybridizes under stringent conditions to a significantly greater extent with total DNA of males of a breed selected from the group consisting of Hereford and Holstein than with total DNA of females of the breed.