Study Of Long Amplication Real-time Quantitative PCR To Detect Zebra Fish DNA Damage

As an endpoint of detecting disease and the toxicity of environmental compounds, DNA damage is always the hot topic of research. It has been reported that long amplicon(above 20 Kb) PCR can reflect the degree of DNA damage basing on the principle that damaged DNA will block the amplification of DNA. In present study, we combined long amplicon PCR technique with real-time quantatative PCR, establish a new method named long-amplication real time PCR( LA-real time PCR). We injected BaP and TBBPA into adμLt male zebrafish throμgh abdominal cavity and exposed zebrafish embryos in vitro, DNA from different tissues of adμLt fish and embryos was isolated and mtDNA as well as nuicleus DNA damages were investigated by LA-real time PCR. The morphological parameters of embryo development including cardiac rate, hatchability and abanormality also were recorded. Meanwhile, we exposed isolated DNA to UV radiation, the mtDNA and nucleus DNA damages also were detected by LA-real time PCR. The resμLts are as follows:(1) UV radiation experiment sμggests that when DNA suffers from physical facors as UV irradiation, it can cause DNA damage, and the extent of the damage has obvious dose effect with the intensity of UV exposure, namely the greater the uv intensity, the greater the degree of DNA damage. Moreover, when the uv intensity reaches 40 J/M2, there is rarely intact DNA chains remained, no amplicon can be amplified, which indicate that that LA-real time PCR can be used to determine the extent of DNA damage.(2) AdμLt zebrafish injection experiment indicates that zebrafish is sensitive to BaP(1, 10 μg/g) and TBBPA(10, 100 μg/g), which can damage DNA in brain, gill, muscle and testis, thereby blocking the PCR amplification.The results show that both BaP and TBBPA can damage mt DNA and nucleus DNA of four tissues. But no obvious dose –effect or time-effect were observed.(3) Embryonic exposure experement indicates that BaP and TBBPA can cause significant damage to the embryos of zebrafish(P < 0.01), and the extent of mtDNA and nucleus DNA damage assiciates with its exposure concentration of TBBPA, while in the Bap group, there has no such relationship. In embryo, the mtDNA is more sensitive to these two chemicals than nucleus DNA.(4) In terms of morphology, BaP and TBBPA exposure induce heart cyst edema, scoliosis and tail hypoplasia etc. in embryo. Embryos are prone to death in early embryonic development stage. The heart showed no significant change at 48 hours of exposure of Bap, but was significantly suppressed at 72 h and 96 h of exposure to 10 μg/L and 20 μg/L, and to 4 μg/L Bap at 96 h. Data from 0.75 mg/L 48 h, 72 h and 96 h, 48 h and 72 h 1.5 mg/L and 5 mg/L48 h group indicated that TBBPA play a significant role in promoting zebrafish embryo heart rate(P < 0.01) instead of restraining.