Detection Of DNA Lesions By DNA Bases Excision Repair And COLD-PCR

DNA lesions is a change in nucleotide sequence in gene duplication process,which can lead to the changes in genetic characteristics.Some studies have shown that the occurrence of many cancers is associated with DNA lesions.Up till the present moment,there are many research on the detection and the repair of DNA lesions.And it is auxiliary effect on molecular diagnosis.COLD-PCR is a new model of PCR,which can selectively amplify the target gene for lesions or mutation with the same reagents and instruments used in conventional PCR reactions.But can make the target gene specific enrichment.In this study,we combine the DNA base repair with COLD-PCR detection technique,established a new method for detecting DNA lesions.The Tm for DNA sequence up to 200 bp in length may vary by approximately 0.2-2℃ and is dependent on the sequence composition.In the process of detecting the DNA oligonucleotide chain containing DNA lesion,we found that effect of this method on gene damage detection was demonstrated in the results of COLD-PCR real-time fluorescence quantitative amplification after base excision repair.And it can achieve the detection of different number of DNA lesions.By optimizing the reaction conditions,we can improve the detection sensitivity.When the damage DNA diluted serially in wild-type DNA at 0.01%,the damage DNA can be detected.The experiment also reduced the concentration of the DNA repair enzyme.And it can improve the detection limit of DNA repair enzyme,which is better than some other methods.We apply this method to cells with DNA lesions,and find that can be able to detect the DNA lesions in cell samples.This method can distinguish different degrees of DNA damage by monitoring the amplification curve of COLD-PCR.We also found some different reactive oxygen species can lead to DNA oxidative damage,which may be a new potential pathway treat cancer by ROS species.In this research,the method of DNA base repair COLD-PCR can detect the DNA lesions.We use the common experimental facilities and the operation is relatively simple.This method can sensitivity detect the DNA lesions,and also detect the DNA lesions in cell.This research is a supporting role for the detection of DNA lesions of cells in vivo,and can be a way to early diagnosis of cancer.