ABSTRACT
Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻⁹ mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 10⁴/mL. When the number of sperm cells was 10³/mL, 10⁴/mL and 10⁵/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.
Subject(s)
Humans , Male , Cell Separation , DNA , Immunomagnetic Separation , Lipocalins , Polymerase Chain Reaction , SpermatozoaABSTRACT
Objective To investigate the 6 short tandem repeats(D2S1338,D8S1179,D16S539,D18S51,D19S433,D21S11) polymorphism in Maonan and Mulao minority and their genetic relationships with other 5 minorities in Guangxi Province and to enrich the genetic database of Maonan and Mulao populations. Methods The allelic frequencies and the genotype of six STR loci were generated from 383 unrelated Maonan and Mulao individuals by PCR_STR and ABI3100. Results Sixty_one and 65 STR alleles were found in the six STR loci of Maonan and Mulao populations,with their frequencies ranging from 0.002 5-0.320 0 and 0.002 7-0.284 2 respectively.Two hundred and sixteen and 218 genotypes were found in Maonan and Mulao populations,with their frequencies ranging from 0.005 0-0.150 0 and 0.005 5-0.158 5 respectively.Their average heterozygosities were above 0.8;polymorphism information content were above 0.8 their accumulative discrimination power were over 0.999 999 98,and the probability of paternity exclusion were over 0.998 3.The results of genetic distance and phylogenetic tree showed that there were closest genetic relationship between Maonan and Mulao minorities,and farthest genetic relationship was existed between Shui and Yi minorities.Seven minorities were clustered to 2 groups on phylogenetic tree.Yi minority was clustered to one group,whereas the other 6 minorities(Maonan,Mulao,Hui,Miao,Shu and Jing nationalities) were clustered to the other group.Conclusion Six STR loci of Maonan and Mulao minorities possesse the characteristic of high genetic diversities which has great practical value.Therefore,the obtained data can be used in human population genetics investigation,individual indentification and paternity test.The genetic variation of STR among 7 populations is basically consistent with their historical culture and geographic distribution.
ABSTRACT
Objective To observe the difference between the bone marrow and peripheral blood engraftment evidence after allogeneic haemopoietic stem cell transplantation(Allo-HSCT) using PCR-STR. Methods DNA from peripheral blood or bone marrow of donors and recipients in different phases were extracted,and 16 STR loci with high polymorphism were amplified by PCR.Separation of the PCR products and fluorescence detection were performed by ABIprism 3100 Genetic Analyzer with capillary electrophoresis.Results The 16 patients included in the study had different levels of engraftment.Twelve patients displayed complete chimerism,while 5 patients showed mixed chimerism.One patient was keeping continuance of remission.The decrease of donor DNA amounts in mixed chimerism was earlier in bone marrow than that in peripheral blood(P