XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
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Posters<br />
27.<br />
SPErm DNA INTEGrITY aSSESMENT USING DIfferENT COMET aSSay<br />
prOTOCOLS<br />
Milena Matejovičová 1 , Michaela Králíková 1 , Jitka Melounová 1 , Martina Vodová 1 ,<br />
Jana Žáková 2 and Igor Crha 2<br />
1<br />
Department of Biochemistry, Medical Faculty MU, Brno, 2 Center for Reproductive<br />
Medicine, Dept. of Obstetrics and Gynecology, Brno<br />
DNA fragmentation in the individual cells can be measured using comet assay. The<br />
method is based on the electrophoresis of nuclear DNA immobilized in agarose gel that<br />
follows after the cell lysis and DNA denaturation. Cells containing damaged DNA have<br />
appearance of the comet: round „head“ representig unfragmented DNA and elongated<br />
„tail“, which intensity and lenght depends on both the extent of DNA fragmentation and<br />
fragment size. Specificity of the comet assay application on sperm consists in particullar<br />
organization of sperm chromatine, which is much more compact than in somatic cells<br />
and it contains a lot of disulfide bonds between DNA and protamines. For assesing DNA<br />
dammage in sperm cells, effective lysis and DNA denaturation without background<br />
damaging effect to DNA are crucial.<br />
A method of modified alkaline/neutral comet assay was used. We studied basal DNA<br />
damage in sperms from subjects undergoing semen analysis in the Center of Assisted<br />
Reproduction (CAR). Semen was liquified for 1 hour at room temperature and 1 ml aliquotes<br />
were frozen in liquid nitrogen. DNA damage level of frozen sperm samples was<br />
compared with the damage in a fresh samples, analyzed on a day of collection. No differences<br />
between fresh and frozen material were found. Next, we compared different<br />
methods of cell lysis. Two different protocol types were applied: 1) proteinase K digestion<br />
during lysis and 2) lithium diodosalicylate (LIS) as a detergent for cell membrane and<br />
chromatine protein core destruction. In both protocols, dithiothreitol (DTT) was used<br />
for disulfide bonds reduction. In the protocol with proteinase K, we have found higher<br />
level of DNA damage when using DTT than without this agent. The least damaged DNA<br />
was observed in assays with LIS and DTT.<br />
144 <strong>XXII</strong>. Biochemistry Congress, Martin
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