Objective: To explore the distribution patterns of the broken sites of the cross-linked DNA by the traditional ultrasonic cleavage method.Methods: A new screening technique “Clone-Sequencing” was developed.By the comparision of clone seqeunce with reference genome, the nucleotide acid sequence of broken site was obtained.The relative cleavage frequency of all 16 dinucleotides was determined by multivariate statistical analysis using the Vector-NTI advance 10 and SPSS PASW Statistics 18.0.Results: By means of the Clone-Sequencing method, 216 clones which occupied different chromosome regions were screened.By the analysis of the DNA sense and anti-sense strand, 432 broken sites were obtained.The statistic analysis of the broken site sequence indicated that the cross-linked DNA was not cleaved randomly along the DNA strands but preferentially occurred in the middle of 5’-ApN-3’ dinucleotides.Addtionally, the relative cleavage frequency decreased in the row of d(ApN)＞＞d(GpN)＞d(CpN)＞d(TpN).Conclusion: The cleavage of cross-linked DNA by the traditional ultrasonic method was not random but sequence-dependent.This results indicated that the present data analysis model of widely used ChIP-Chip or ChlP-Seq which involved the sonication clevage of cross-linked DNA need to be reevaluated.A new strategy which could titrate the sequence bias of sonication cleavage need to develope.%目的:研究在交联状态下超声方法处理DNA是否导致DNA随机断裂.方法:通过建立一种新的Clone-Sequencing筛选技术,利用NCBI等相关网站,结合Vector-NTI等软件分析得到不同断裂位点的核苷酸序列,利用SPSS 18.0软件,对不同断裂位点核苷酸序列的分布进行统计学分析.结果:利用Clone-Sequencing技术得到216个来源于不同染色体位置的克隆片段,通过对其正义链和反义链的5'-DNA断裂末端的分析,得到432个超声断裂位点的核苷酸序列信息.对断裂位点核苷酸序列进行统计分析发现,超声处理后,交联DNA的断裂位点并不像过去认为的完全随机分布,而是存在明显的偏性,即对于所有16种不同的二核苷酸组合,超声处理导致交联DNA更易在5'-ApN-3'寡核苷酸中间的磷酸二醋健处断裂,且其相对断裂频率呈现d(ApN＞＞d(GpN)＞d(CpN)＞d(TpN)的趋势.结论:传统的超声方法处理,交联 DNA断裂位点并不是完全随机分布,而是存在着明显的序列偏好性,这一新发现提示,对于目前广泛使用的、涉及交联DNA超声处理的ChIP-Chip或ChIP-Seq等高通量及其衍生技术结果的统计分析,需要引入更加合理有效的分析策略.
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