[논문]Evaluation of DNA Fragments on Boar Sperm by Ligation-mediated Quantitative Real Time PCR

Lee, Eun-Soo; Choi, Sun-Gyu; Yang, Jae-Hun; Bae, Mun-Sook; Park, Jin-Young; Park, Hong-Min; Han, Tae-Kyu; Hwang, You-Jin; Kim, Dae-Young

Evaluation of DNA Fragments on Boar Sperm by Ligation-mediated Quantitative Real Time PCR 원문보기

Journal of embryo transfer = 한국수정란이식학회지, v.25 no.2, 2010년, pp.111 - 116

Lee, Eun-Soo (Division of Biological Science, Gachon University of Medicine and Science) , Choi, Sun-Gyu (Division of Biological Science, Gachon University of Medicine and Science) , Yang, Jae-Hun (Division of Biological Science, Gachon University of Medicine and Science) , Bae, Mun-Sook (Konkuk University School of Medicine) , Park, Jin-Young (Division of Biological Science, Gachon University of Medicine and Science) , Park, Hong-Min (Division of Biological Science, Gachon University of Medicine and Science) , Han, Tae-Kyu (Division of Biological Science, Gachon University of Medicine and Science) , Hwang, You-Jin (Division of Biological Science, Gachon University of Medicine and Science) , Kim, Dae-Young (Division of Biological Science, Gachon University of Medicine and Science)

Abstract ▼ AI-Helper

Sperm chromatin integrity is essential for successful fertilization and development of an embryo. Reported here is a quantification of DNA fragments which is intimately associated with reproductive potential to provide one of criteria for sperm chromatin integrity. Three sperm populations were considered: CONTROL (no treatment), UV irradiation (48mW/$cm^2$, 1h) and $H_2O_2$ (oxidative stress induced by hydrogen peroxide, 10 mM, 50 mM and 100 mM). DNA fragments in boar sperm were evaluated by using ligation-mediated quantitative real-time polymerase chain reaction (LM-qPCR) assay, which relies on real-time qPCR to provide a measure of blunt 5' phosphorylated double strand breaks in genomic DNA. The results in agarose gel electrophoresis showed no significant DNA fragmentation and no dose-dependent response to $H_2O_2$. However, the remarkable difference in shape and position was observed in melting curve of LM-qPCR. This result supported that the melting curve analysis of LM-qPCR presented here, could be more sensitive and accurate than previous DNA fragmentation assay method.

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문제 정의

The LM~qPCR is unified Method of Ligation-Mediated PCR and Real Time PCR. The LMqPCR has a merit that products are more accurate identified than previous method by melting curve analysis The purpose of this study was to evaluate the efficiency of LMqPCR assay when used in boar sperm samples with different conditions.

제안 방법

Real-time PCR was performed using an iQ™5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories) which monitored the PCR reactions, for two sets of primers simultaneously, and produced separate fluorescence amplification plots for each primer set product. The quantification was performed by experimental determination of the threshold cycle (Ct), defined as the PCR cycle number.
The procedure was adapted from a previously described method (Staley et al., 1997; Salas et al, 2007) and modified for this study. Genomic DNA of each sample was mixed with 0.
for each primer set product. The quantification was performed by experimental determination of the threshold cycle (Ct), defined as the PCR cycle number. The quantities relative to control sample were automatically estimated using the iQ™5 Optical System Software.

성능/효과

In addition, the decline of fluorescence was likely to HsOs-dose-dependent. From these results, we concluded the melting curve analysis of LM-qPCR products is more accurate and sensitive method than previous DNA fragmentation assay.
However the nucleosomal ladders in size 200—400 bp appeared in agarose gel electrophoresis for LM-qPCR because LM-qPCR can specifically amplified 5' phosphorylated blunt DNA ends. We could confirm that condition of DNA fragmentation among each sperm samples is different, but the LM-qPCR result was contrast to those we had expected. We expected that nucleosomal fragements were observed in dose dependent manner, but the result of agarose gel electrophoresis was completely opposed.

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