Methods and kits for isolating sperm cells

Abstract

Disclosed are methods for isolating sperm cells from an aqueous sample and kits for isolating sperm cells from an aqueous sample.

1 . A method of separating sperm cells from an aqueous sample comprising:
 (a) contacting the aqueous sample with a non-aqueous liquid having a density greater than about 1.00 g/cm 3 , wherein the density of the non-aqueous liquid is sufficiently low to permit pelleting of at least a portion of the sperm cells in the sample;   (b) applying a force to the contacted sample for a period of time sufficient to form an aqueous layer, a non-aqueous layer, and a sperm pellet, thus separating sperm cells from the aqueous sample.   
     
     
         2 . The method of  claim 1 , wherein the force is applied by centrifugation. 
     
     
         3 . The method of  claim 1 , wherein the non-aqueous liquid has a density of at least about 1.01 g/cm 3 . 
     
     
         4 . The method of  claim 1 , wherein the non-aqueous liquid has a density of about 1.29 g/cm 3  or lower. 
     
     
         5 . The method of  claim 1 , wherein the non-aqueous liquid comprises chloroform. 
     
     
         6 . The method of  claim 1 , wherein the non-aqueous liquid has a density in the range of from about 1.02 g/cm 3  to about 1.29 g/cm 3 . 
     
     
         7 . The method of  claim 1 , wherein the non-aqueous liquid has a density in the range of from about 1.050 g/cm 3  to about 1.058 g/cm 3 . 
     
     
         8 . The method of  claim 1 , wherein the aqueous sample comprises lysed epithelial cells. 
     
     
         9 . The method of  claim 1 , further comprising the step of:
 (c) removing the aqueous layer.   
     
     
         10 . The method of  claim 9 , further comprising the step of:
 (d) contacting the non-aqueous layer and sperm pellet with a chaotropic agent.   
     
     
         11 . The method of  claim 10 , wherein the chaotropic agent comprises a chaotropic salt. 
     
     
         12 . The method of  claim 10 , wherein the chaotropic agent comprises a detergent, and further comprising the step of:
 (e) contacting the non-aqueous layer and sperm cell pellet of step (d) with phenol:chloroform.   
     
     
         13 . The method of  claim 9 , further comprising the step of:
 (d) removing the non-aqueous layer.   
     
     
         14 . The method of  claim 13 , further comprising the step of:
 (e) contacting the cell pellet of step (d) with an aqueous detergent and phenol:chloroform.   
     
     
         15 . A method of isolating DNA from sperm cells in an aqueous sample comprising lysed epithelial cells comprising:
 (a) contacting the aqueous sample with a non-aqueous liquid having a density of greater than about 1.00 g/cm 3 , wherein the density of the non-aqueous liquid is sufficiently low to permit pelleting of at least a portion of the sperm cells in the sample;   (b) centrifuging the contacted sample to form an aqueous layer, a non-aqueous layer, and a sperm cell pellet;   (c) removing the aqueous layer;   (d) contacting the non-aqueous layer and sperm cell pellet with lysis buffer comprising a chaotropic agent and a reducing agent; and   (e) isolating DNA from the contacted sperm cell pellet of step (d).   
     
     
         16 . A method of isolating DNA from sperm cells in an aqueous sample comprising lysed epithelial cells comprising:
 (a) contacting the aqueous sample with a non-aqueous liquid having a density of greater than about 1.00 g/cm 3 , wherein the density of the non-aqueous liquid is sufficiently low to permit pelleting of at least a portion of the sperm cells in the sample;   (b) centrifuging the contacted sample to form an aqueous layer, a non-aqueous layer, and a sperm cell pellet;   (c) removing the aqueous layer and non-aqueous layer;   (d) contacting the sperm cell pellet with an aqueous solution comprising a detergent and phenol:chloroform; and   (e) isolating DNA from the contacted sperm cell pellet of step (d).   
     
     
         17 . A kit for isolating sperm cells from a sample comprising sperm cells, comprising a non-aqueous liquid having a density of greater than about 1.00 g/cm 3 , wherein the density of the non-aqueous liquid is sufficiently low to permit pelleting of at least a portion of the sperm cells in the sample. 
     
     
         18 . The kit of  claim 17 , further comprising an aqueous buffer. 
     
     
         19 . The kit of  claim 17 , further comprising Proteinase K. 
     
     
         20 . The kit of  claim 17 , further comprising a chaotropic agent. 
     
     
         21 . The kit of  claim 17 , further comprising a spin basket.