Kometová analýza jako nástroj pro stanovení integrity DNA spermií. Porovnání různých protokolů.
| Title in English | Comet assay as a tool for assesment sperm DNA integrity. Comparison of different protocols. |
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| Year of publication | 2009 |
| Type | Conference abstract |
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| Description | Comet assay is a method for measuring the amount of DNA fragmentation in the individual cells. Cells containing damaged DNA have appearance of a comet: round „head“ representing unfragmented DNA and elongated „tail“, intensity and lenght of which depends on both, the extent of DNA fragmentation and fragment size. Specificity of the comet assay application on sperm consists in particullar organization of sperm chromatine, which is 6 fold more compact than in somatic cells and it contains a lot of disulfide bonds in protamines. In the present work, we studied basal DNA damage in sperms collected from subjects undergoing semen analysis in the Assisted Reproduction Center. DNA damage of frozen sperm samples was compared with level of DNA damage in fresh samples analyzed during a day of collection. We have not found a differences in DNA damage between fresh and frozen material. Further, we have compared 2 different methods of cell lysis. Protocol A included proteinase K digestion during lysis and protocol B used lithium diodosalicylate as a main detergent for cell membrane and chromatine protein core destruction. In both protocols, dithiothreitol (DTT) was used for disulfide bonds reduction. However, in the protocol A with proteinase K, we have found higher level of DNA damage when using DTT than without this agent. The least damaged DNA was observed in assays when using the protocol B. |
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