Selective Lysis of Sperm Cells
The teaching provides a method for selectively lysing sperm cells in a mixed cell sample, particularly in the field of forensic sciences. Reagents and kits for carrying out the methods are also provided.
1 . A method of selectively lysing a sperm cell in a mixture of at least one type of non-sperm cell comprises:
i) incubating the mixture with the selective sperm lysis buffer, wherein the buffer comprises at least on disulfide bond reducing reagent and at least one salt reagent, wherein the salt reagent is at a concentration to lyse a sperm cell whereas a non-sperm cell is intact.
ii) lysing the sperm cell, while the non-sperm cell is intact, and
iii) separating the non-sperm cell from the lysed sperm cell.
2 . The method of claim 1 , wherein at least one type of non-sperm cells comprises erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, tumor cells, neurons, glial cells, astrocytes, red blood cells, white blood cells, macrophages, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, antigen presenting cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, prostate cells, vaginal epithelial cells, testicular cells, sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, squamous epithelial cells, osteocytes, osteoblasts, or osteoclasts.
3 . The method of claim 1 , wherein at least one disulfide bond reducing agent comprises DTT, TCEP, ME or GSH.
4 . The method of claim 1 , wherein the concentration of at least one salt reagent is at least 0.1M.
5 . The method of claim 1 , wherein the concentration of at least one salt reagent is at least 0.25M.
6 . The method of claim 4 , wherein the salt comprises at least LiCl, NaCl, KCl. Li 2 SO 4 , Na 2 SO 4 , K 2 SO 4 , MgCl 2 , CaCl 2 , MgSO 4 , CaSO 4 , NaNO 3 , KNO 3 , Mg(NO 3 ) 2 , or Ca(NO 3 ) 2 .
7 . The method of claim 5 , wherein the salt comprises at least NaCl, KCl or MgCl 2 .
8 . A method of differential extraction of sperm cell DNA from a cell mixture from a sample carrier, wherein the cell mixture comprises a sperm cell and at least one type of non-sperm cell, comprises:
i) washing the cell mixture with washing solution and remove the supernatant,
ii) incubating the mixture with the selective sperm lysis buffer, wherein the buffer comprises at least one disulfide bond reducing reagent and at least one salt reagent, wherein the salt reagent is at a concentration to lyse a sperm cell whereas a non-sperm cell is intact.
ii) lysing the sperm cell, while the non-sperm cell is intact, and
iii) separating the non-sperm cell from the lysed sperm cell.
9 . The method of claim 8 , wherein at least one type of non-sperm cells comprises erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, tumor cells, neurons, glial cells, astrocytes, red blood cells, white blood cells, macrophages, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, antigen presenting cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, prostate cells, vaginal epithelial cells, testicular cells, sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, squamous epithelial cells, osteocytes, osteoblasts, or osteoclasts.
10 . The method of claim 8 , wherein at least one disulfide bond reducing agent comprises DTT, TCEP, ME or GSH.
11 . The method of claim 8 , wherein the concentration of at least one salt reagent is at least 0.1M.
12 . The method of claim 8 , wherein the concentration of at least one salt reagent is at least 0.25M.
13 . The method of claim 11 , wherein the salt comprises at least LiCl, NaCl, KCl. Li 2 SO 4 , Na 2 SO 4 , K 2 SO 4 , MgCl 2 , CaCl 2 , MgSO 4 , CaSO 4 , NaNO 3 , KNO 3 , Mg(NO 3 ) 2 , or Ca(NO 3 ) 2 .
14 . The method of claim 12 , wherein the salt comprises at least NaCl, KCl or MgCl 2 .
15 . A kit for isolating sperm DNA from non-sperm cell DNA comprises (i) a selective sperm lysis buffer comprising at least one disulfide bond reducing agent and at least one salt, wherein the salt is at the concentration wherein the sperm cell is lysed while the other cells are intact.
16 . The kit of claim 15 , where the salt concentration in the selective sperm lysis buffer is at least 0.1M.
17 . The kit of claim 16 , wherein the salt is comprises at least LiCl, NaCl, KCl. Li 2 SO 4 , Na 2 SO 4 , K 2 SO 4 , MgCl 2 , CaCl 2 , MgSO 4 , CaSO 4 , NaNO 3 , KNO 3 , Mg(NO 3 ) 2 , or Ca(NO 3 ) 2 .
18 . The kit of claim 15 , wherein the concentration of at least one salt reagent is at least 0.25M.
19 . The kit of claim 18 , wherein the salt reagent comprises at least NaCl, KCl or MgCl 2 .
20 . The kit of claim 15 , wherein at least one disulfide bond reducing agent comprises at least DTT, TCEP, ME or GSH.
21 . The kit of claim 20 , where the salt concentration in the selective sperm lysis buffer is at least 0.1M.
22 . The kit of claim 21 , wherein the salt comprises at least LiCl, NaCl, KCl. Li 2 SO 4 , Na 2 SO 4 , K 2 SO 4 , MgCl 2 , CaCl 2 , MgSO 4 , CaSO 4 , NaNO 3 , KNO 3 , Mg(NO 3 ) 2 , or Ca(NO 3 ) 2 .
23 . The kit of claim 20 , wherein the concentration of at least one salt reagent is at least 0.5M.
24 . The kit of claim 23 , wherein the salt reagent comprises at least NaCl, KCl or MgCl 2 .