Methods and kits for capturing sperm nucleic acids

Patent metadata

Field	Value
Publication number	US-10030241-B2
Application number	US-201615150806-A
Country	US
Kind code	B2
Filing date	May 10, 2016
Priority date	Mar 30, 2015
Publication date	Jul 24, 2018
Grant date	Jul 24, 2018

Abstract

The invention generally relates to methods and kits for capturing sperm nucleic acids from or in a biological sample. In one embodiment the method the method includes, contacting the sample with a lysis solution, having a protamine-DNA complex, to lyse the cell and applying a protamine-specific antibody. This results in the protamine-specific antibody binding to the protamine-DNA to form a complex which may be captured, purified, or detected. Also provided are kits for carrying out the disclosed methods.

First claim

What is claimed is:

1. A method of capturing a sperm deoxyribo nucleic acid (DNA) in a biological sample, comprising:
contacting a lysis solution with the biological sample, where the biological sample comprises at least a sperm cell or a sperm cell lysate, and the lysis solution comprises a protamine-DNA complex resulting in a lysed sperm cell, wherein the lysis solution comprises at least one of a reducing agent and a detergent;
applying a sequestration agent after the sperm cell lysis to sequester the detergent of the lysis solution;
applying at least a protamine-specific antibody to the lysed sperm cell after the sequestration of the detergent, wherein the protamine-specific antibody binds to the protamine-DNA complex of the lysed sperm cell to form an antibody-protamine-DNA complex;
capturing the antibody-protamine-DNA complex; and
optionally, detecting the sperm DNA from the captured antibody-protamine-DNA complex.

2. The method of claim 1 , further comprising removing or sequestering the lysis solution prior to applying the protamine-specific antibody.

3. The method of claim 1 , wherein the lysis solution comprises a reducing agent selected from dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) or combination thereof.

4. The method of claim 1 , wherein the lysis solution comprises a detergent selected from sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS) or sarkosyl.

5. The method of claim 1 , wherein the lysis solution comprises 40 mM dithiothreitol (DTT), 0.5% to 2% sodium dodecyl sulfate (SDS) or combinations thereof.

6. The method of claim 1 , wherein the sequestration agent comprises ligand-activated core beads coated with size exclusion shell, alpha-cyclodextrin, size exclusion resin or combinations thereof.

7. The method of claim 1 , wherein the protamine-specific antibody binds to the protamine DNA complex during incubation in a range from 4° C. to 37° C.

8. The method of claim 1 , wherein the protamine-specific antibody binds to the protamine DNA complex during incubation at 4° C. for a time in a range from 10 minutes to 4 hrs.

9. The method of claim 1 , wherein the capturing of the antibody-protamine-DNA complex is achieved by a capturing agent.

10. The method of claim 9 , wherein the capturing of the antibody-protamine-DNA complex is achieved by pre-coupling the capturing agent with the protamine-specific antibody or by separately adding the capturing agent such that the capturing agent binds to the protamine-specific antibody.

11. The method of claim 9 , wherein the capturing agent comprises a secondary antibody, agarose beads, paramagnetic beads, protein A, streptavidin, sephadex, glass bead, cellulose, nitrocellulose, quartz, a lateral flow strip or combinations thereof.

12. The method of claim 11 , wherein the capturing agent is a secondary antibody specific to the protamine-specific antibody.

13. The method of claim 9 , wherein the captured antibody-protamine-DNA complex is washed to remove unbound material while retaining the protamine-DNA complex.

14. The method of claim 13 , further comprising incubating the captured antibody-protamine-DNA complex in an ion exchange resin.

15. The method of claim 14 , wherein the captured antibody-protamine-DNA complex is incubated at 95° C. for at least 10 minutes.

16. The method of claim 1 , wherein a reporter moiety is coupled to the protamine-specific antibody and wherein the detection of the reporter moiety indicates the presence of sperm DNA in the biological sample.

17. The method of claim 16 , wherein the reporter moiety comprises a chromophore moiety, a fluorescent moiety, a phosphorescence moiety, an affinity probe, a magnetic probe, a paramagnetic probe, a metallic probe or combinations thereof.

18. The method of claim 1 , wherein the detection of the sperm DNA comprises one or more amplification reactions of the sperm DNA to form an amplified DNA.

19. The method of claim 18 , further comprising analyzing the amplified DNA.

20. The method of claim 1 , wherein the biological sample further comprises epithelial cells, somatic cells, blood cells, or combinations thereof.

21. The method of claim 1 , wherein the biological sample is selected from a forensic sample comprising sperm cells.

22. The method of claim 1 , wherein the biological sample is affixed to a solid substrate.

23. The method of claim 1 , wherein the biological sample is acquired at least three days post ejaculation.

24. A method of capturing sperm deoxyribonucleic acid (DNA) in a biological sample, comprising:
providing the biological sample comprising at least a sperm cell, a partially lysed sperm cell or a sperm cell lysate, wherein the sperm cell, partially lysed sperm cell or sperm cell lysate comprises a protamine-DNA complex;
contacting a lysis solution to the sample to lyse the sperm cell or partially lysed sperm cell, wherein the lysis solution comprises at least one of a reducing agent and a detergent;
applying a sequestration agent after the sperm cell lysis to sequester the detergent of the lysis solution;
applying at least a protamine-specific antibody to the lysed sperm cell after the sequestration of the detergent, wherein the protamine-specific antibody binds to the protamine-DNA complex of the lysed sperm cell to form an antibody-protamine-DNA complex;
capturing the antibody-protamine-DNA complex by adding a capturing agent; and
optionally, detecting the sperm DNA from the captured antibody-protamine-DNA complex by a DNA amplification reaction.

25. The method of claim 24 , wherein the biological sample is affixed to a solid substrate.

26. The method of claim 24 wherein the biological sample is acquired at least three days post ejaculation.

27. A method of purifying deoxyribonucleic acid (DNA) from a biological sample, comprising:
providing the sample comprising a protamine-DNA complex and at least one of a reducing agent and a detergent;
applying a sequestration agent to the protamine-DNA complex to sequester the detergent of the protamine-DNA complex;
applying at least a protamine-specific antibody to the protamine-DNA complex after the sequestration of the detergent to form an antibody-protamine-DNA complex;
capturing the antibody-protamine-DNA complex using a capturing agent; and
purifying DNA from the captured antibody-protamine-DNA complex.

28. The method of claim 27 wherein the biological sample is affixed to a solid substrate.

29. A method of claim 1 , wherein the biological sample is acquired at least three days post ejaculation.

30. A kit for capturing a sperm deoxyribonucleic acid (DNA) in a biological sample comprising:
at least one of a reducing agent, a detergent, or a combination thereof;
a sequestering agent to sequester the detergent;
a protamine specific antibody;
a capture agent; and
a lysis buffer.

31. The kit of claim 30 wherein the capturing agent comprises a lateral flow strip, a magnetic bead, agarose beads or an affinity column.

32. The kit of claim 31 wherein the capturing agent is a lateral flow strip.

33. The kit of claim 30 wherein the sequestration agent comprises ligand-activated core beads coated with size exclusion shell, alpha-cyclodextrin, size exclusion resin or combinations thereof.

Assignees

• Gen Electric

Inventors

• Spooner Patrick Mccoy
• Tatnell Peter James
• Horton Jeffrey Kenneth
• Nelson John Richard
• Gerdes Michael John
• Kiel Suzana
• Lenigk Ralf
• Schenk Alexander
• Sun Wei

Classifications

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12Q2600/124

  Animal traits, i.e. production traits, including athletic performance or the like · CPC title

• G01N27/44743

  Introducing samples · CPC title

• G01N33/689

  related to pregnancy or the gonads · CPC title

• C12N15/1013 Primary

  by using magnetic beads · CPC title