为建立多态性高、稳定性好的洋葱RAPD-PCR反应体系,采用正交设计,研究了Taq酶、Mg2+、引物和dNTP 4种RAPD-PCR反应组分浓度变化对扩增结果的影响,在此基础上对模板DNA用量、扩增程序中退火温度和反应循环次数进行了筛选。试验结果表明,...为建立多态性高、稳定性好的洋葱RAPD-PCR反应体系,采用正交设计,研究了Taq酶、Mg2+、引物和dNTP 4种RAPD-PCR反应组分浓度变化对扩增结果的影响,在此基础上对模板DNA用量、扩增程序中退火温度和反应循环次数进行了筛选。试验结果表明,洋葱20μl RAPD-PCR优化反应体系为1×Buffer、2.0 mmo1/L Mg2+、1.0 UTaqDNA聚合酶、200μmo1/L dNTP、0.6μmo1/L引物、2%甘油和15 ng DNA模板;PCR扩增程序为94℃预变性4 m in;94℃变性30 s,35℃退火40 s,72℃延长1.5 m in,45个循环;72℃保温延伸7 m in。展开更多
The lethal and sublethal effects of oils on aquatic organisms have been widely investigated, but the potential molecular impacts of oils on aquatic organisms are remaining unclear now. In order to realize the effects ...The lethal and sublethal effects of oils on aquatic organisms have been widely investigated, but the potential molecular impacts of oils on aquatic organisms are remaining unclear now. In order to realize the effects of diesel oil on the Zhe oyster, the RAPD （random amplified polymorphic DNA） technique was used. RAPD is a useful assay procedure for the detection of genotoxin-induced DNA damage and mutations. In the present study, the Zhe oysters were exposed to diesel oil at different concentrations and for different exposure periods. The results showed that the DNA band change in RAPD profiles of oysters following diesel oil treatment included loss of normal DNA bands, the appearance of new DNA bands and variations in DNA intensity compared to oysters not exposed to diesel oil. The effects of changes to GTS （genome template stability） were time- and concentration-dependent, the GTS of 10 mg/L was 82.46%, 80.70% and 63.15% in the 8, 16 and 32 days, the GTS of 20 mg/L was 75.44%, 71.93% and 56.14% in the 8, 16 and 32 days, the GTS of 40 mg/L was 73.68%, 70.18% and 56.14% in the 8, 16 and 32 days, respectively. The DNA polymorphisms detected by RAPD analysis could be used as a useful biomarker assay for the detection of genotoxic effects in diesel oil pollution on the oysters, and may be useful for environmental contamination risk assessment.展开更多
文摘为建立多态性高、稳定性好的洋葱RAPD-PCR反应体系,采用正交设计,研究了Taq酶、Mg2+、引物和dNTP 4种RAPD-PCR反应组分浓度变化对扩增结果的影响,在此基础上对模板DNA用量、扩增程序中退火温度和反应循环次数进行了筛选。试验结果表明,洋葱20μl RAPD-PCR优化反应体系为1×Buffer、2.0 mmo1/L Mg2+、1.0 UTaqDNA聚合酶、200μmo1/L dNTP、0.6μmo1/L引物、2%甘油和15 ng DNA模板;PCR扩增程序为94℃预变性4 m in;94℃变性30 s,35℃退火40 s,72℃延长1.5 m in,45个循环;72℃保温延伸7 m in。
文摘The lethal and sublethal effects of oils on aquatic organisms have been widely investigated, but the potential molecular impacts of oils on aquatic organisms are remaining unclear now. In order to realize the effects of diesel oil on the Zhe oyster, the RAPD （random amplified polymorphic DNA） technique was used. RAPD is a useful assay procedure for the detection of genotoxin-induced DNA damage and mutations. In the present study, the Zhe oysters were exposed to diesel oil at different concentrations and for different exposure periods. The results showed that the DNA band change in RAPD profiles of oysters following diesel oil treatment included loss of normal DNA bands, the appearance of new DNA bands and variations in DNA intensity compared to oysters not exposed to diesel oil. The effects of changes to GTS （genome template stability） were time- and concentration-dependent, the GTS of 10 mg/L was 82.46%, 80.70% and 63.15% in the 8, 16 and 32 days, the GTS of 20 mg/L was 75.44%, 71.93% and 56.14% in the 8, 16 and 32 days, the GTS of 40 mg/L was 73.68%, 70.18% and 56.14% in the 8, 16 and 32 days, respectively. The DNA polymorphisms detected by RAPD analysis could be used as a useful biomarker assay for the detection of genotoxic effects in diesel oil pollution on the oysters, and may be useful for environmental contamination risk assessment.
