Methods and systems for assessing and/or quantifying sperm cell subpopulations bearing a specific genetic signature
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.
1. 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;
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; and
a third oligonucleotide agent that selectively binds to an autosomal chromosome nucleic acid sequence.
2. The method of claim 1 , further comprising isolating sperm DNA from a population of sperm cells from a semen sample.
3. The method of claim 2 , further comprising removing laser ablated sperm cells from said semen sample of sperm cells prior to said isolating sperm DNA.
4. The method of claim 1 , wherein said population of cells comprise an X-skewed sperm population.
5. The method of claim 1 , wherein said step of detecting comprises amplifying at least one of an X-chromosome tamet site, a Y-chromosome target site, and a housekeeping target site.
6. The method of claim 1 , wherein the first oligonucleotide agent that selectively binds to an X-chromosome nucleic acid sequence comprises:
a first primer;
a second primer; and
a probe.
7. The method of claim 1 , wherein the second oligonucleotide agent that selectively binds to a Y-chromosome nucleic acid sequence comprises:
a first primer;
a second primer; and
a probe.
8. The method of claim 7 , wherein said X-chromosome primer sequences comprise SEQ ID NOs: 4 and 5, and said probe sequence comprises SEQ ID NO: 6.
9. The method of claim 8 , wherein said Y-chromosome primer sequences comprise SEQ ID NOs: 7 and 8, and said probe sequence comprises SEQ ID NO: 9.
10. The method of claim 7 , wherein said probe includes a detectable label.
11. The method of claim 8 , wherein said probe includes a detectable label.
12. The method of claim 1 , wherein said third oligonucleotide agent comprises primer sequences comprising SEQ ID NOs: 1 and 2, and a probe sequence comprising SEQ ID NO: 3.
13. A kit 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;
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; and
a third oligonucleotide agent that selectively binds to an autosomal chromosome nucleic acid sequence.
14. The kit of claim 13 , wherein:
said first nucleotide agent comprises oligonucleotide primer sequences SEQ ID NOs: 4 and 5, and oligonucleotide probe sequence SEQ ID NO: 6;
said second nucleotide agent comprises oligonucleotide primer sequences SEQ ID NOs:7 and 8, and oligonucleotide probe sequence SEQ ID NO: 9; and
said third nucleotide agent comprises oligonucleotide primer sequences SEQ ID NOs: 1 and 2, and oligonucleotide probe sequence SEQ ID NO: 3.
15. The kit of claim 13 , further comprising reagents and buffers for removing non-viable sperm cells.
16. The kit of claim 13 , further comprising reagents and buffers for isolating DNA from a sample of sperm cells.
17. The method of claim 1 , wherein said autosomal chromosome nucleic acid sequence is a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) nucleotide sequence.
18. The method of claim 2 , wherein said semen sample is from Bos Taurus, Bos indicus, Bos bubalis, or hybrids thereof.
19. The method of claim 1 , wherein said detecting comprises performing multiplex ddPCR.
20. 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 SEQ ID NOs: 4 and 5, and a probe sequence comprising SEQ ID NO: 6;
a second oligonucleotide agent that selectively binds to a Y-chromosome nucleic acid sequence comprising SEQ ID NOs: 7 and 8, and a probe sequence comprising SEQ ID NO: 9; and
a third oligonucleotide agent that selectively binds to an autosomal chromosome nucleic acid sequence wherein said detecting comprises performing multiplex ddPCR.