Methods of capturing sperm nucleic acids

Patent metadata

Field	Value
Publication number	US-2016289735-A1
Application number	US-201514672358-A
Country	US
Kind code	A1
Filing date	Mar 30, 2015
Priority date	Mar 30, 2015
Publication date	Oct 6, 2016
Grant date	—

Abstract

A method is provided herein, wherein the method of capturing a sperm deoxyribo nucleic acid (DNA) in a sample, comprises contacting a lysis solution to the sample comprising at least a sperm cell or a sperm cell lysate to lyse the sperm cell. The sperm cell or sperm cell lysate comprises a protamine-DNA complex. The method further comprises applying at least a protamine-specific antibody to the lysed sperm cell, wherein the protamine-specific antibody binds to the protamine-DNA complex of the lysed sperm cell to form an antibody-protamine-DNA complex. The antibody binding is followed by capturing the antibody-protamine-DNA complex; and isolating and detecting the sperm DNA from the captured antibody-protamine-DNA complex.

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 to the biological sample comprising at least a sperm cell or a sperm cell lysate comprising a protamine-DNA complex to lyse the sperm cell; applying at least a protamine-specific antibody to the lysed sperm cell, 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 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, a detergent or combination thereof.

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

5 . The method of claim 3 , wherein the lysis solution comprises a detergent selected from sodium dodecyl sulfate (SDS).

6 . The method of claim 3 , wherein the lysis solution comprises 40 mM dithiothreitol (DTT), 0.5% to 2% sodium dodecyl sulfate (SDS) or combinations thereof.

7 . The method of claim 3 , further comprising applying a sequestration agent after cell lysis to sequester detergent prior to applying the protamine-specific antibody to the lysed sperm cell.

8 . The method of claim 7 , wherein the sequestration agent comprises ligand-activated core beads coated with size exclusion shell, alpha-cyclodextrin, size exclusion resin or combinations thereof.

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

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

11 . The method of claim 1 , wherein the capturing of the antibody-protamine-DNA complex is achieved by a capturing agent, wherein the capturing agent is pre-coupled with the protamine-specific antibody.

12 . The method of claim 11 , wherein the capturing agent comprises a secondary antibody, agarose beads, paramagnetic beads, protein A, streptavidin, sephadex, glass bead or combinations thereof.

13 . The method of claim 1 , wherein the capturing of the antibody-protamine-DNA complex is achieved by further adding a capturing agent, wherein the capturing agent binds to the protamine-specific antibody.

14 . The method of claim 13 , wherein the capturing agent comprises a secondary antibody, agarose beads, paramagnetic beads, protein A, streptavidin, sephadex, glass bead or combinations thereof.

15 . The method of claim 13 , wherein the capturing agent is a secondary antibody specific to the protamine-specific antibody.

16 . The method of claim 13 , wherein the captured antibody-protamine-DNA complex is washed to remove unbound material and purify the sperm DNA.

17 . The method of claim 16 , further comprising incubating the captured antibody-protamine-DNA complex in an ion exchange resin.

18 . The method of claim 17 , wherein the captured antibody-protamine-DNA complex is incubated at about 95° C. for at least about 10 minutes.

19 . 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 sample.

20 . The method of claim 19 , 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.

21 . The method of claim 1 , wherein the detection of the sperm DNA comprises one or more amplification reactions of the sperm DNA.

22 . The method of claim 21 , further comprising analyzing the amplified DNA.

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

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

25 . 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; applying at least a protamine-specific antibody to the lysed sperm cell, 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 detecting the sperm DNA from the captured antibody-protamine-DNA complex by a DNA amplification reaction.

26 . A method of purifying deoxyribo nucleic acid (DNA) from a biological sample, comprising:
providing the sample comprising a protamine-DNA complex; applying at least a protamine-specific antibody to the protamine-DNA complex 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.

Assignees

• Gen Electric

Inventors

• Gerdes Michael John
• Nelson John Richard
• Spooner Patrick Mccoy
• Lenigk Ralf

Classifications

• C12N15/1003

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• C12Q1/6804 Primary

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

• C12Q2522/101

  Single or double stranded nucleic acid binding proteins · CPC title

• G01N33/689

  related to pregnancy or the gonads · CPC title