四核苷酸重复序列单链扩增特性及机理探究

简单重复序列广泛存在于多种生物基因组中,其生物学意义越来越受到人们的重视.许多简单重复序列易于扩增变长,某些重复序列的异常延伸是造成一些遗传疾病的直接原因.本研究以20 nt的60种四重复和6种二重复序列单链为模板,系统研究了它们在嗜热DNA聚合酶作用下等温扩增的特点.电泳结果显示,多数单链模板能扩增变长,即使链内没有互补碱基的序列也可被扩增,如(AGGA)5.定量分析结果显示:回文序列扩增最快;二重复序列比相同碱基组成的四重复序列有更宽的适于扩增的温度范围;G和C含量多的DNA较G和C含量少的序列更易扩增,而且G和C含量越多越适于在较高的温度下扩增;重复单位含两相同嘧啶的链多数比其互补链更易扩增;产物浓度与时间基本呈线性关系.限制性酶切产物结果显示,扩增产物与模板具有相同的重复单位,是重复序列的简单延伸.最后,根据实验结果和相关文献,提出了包括链内滑动扩增和发卡DNA介导扩增两阶段的重复序列单链扩增模型,以对重复序列非特异扩增和相关疾病发生机制的研究提供参考.

Amplification Characteristics of Single-strand Tetranucleotide Repetitive Sequences and Its Mechanism

Simple sequence repeats(SSR), whose biological significance causes people's increasing attention, are widely distributed in genomes of many organisms. Many of them can be elongated easily and abnormal extension can directly result in certain hereditary diseases in some cases. In this research, sixty kinds of tetranucleotide repetitive sequences (TRS) and six kinds of dinucleotide repetitive sequences (DRS) of 20 nt single strands were used for isothermal amplification by thermophilic DNA polymerase. The electrophoresis results demonstrated that most of single-strand repeats, even the sequences with no complementary bases inside like AGGA, can be elongated. The results of quantitative analysis demonstrated: palindromic sequences were amplified most easily; DRS could be amplified at a broader range of temperature than TRS; DNA with more G and C, were more suitable for amplification under higher temperature; Most strands whose repetitive unit contains two same pyrimidines were amplified more easily than their complimentary ones; the concentration of products exhibited linear relationship with time. The results of restriction endonuclease digestion indicated that the products had the same repetitive unit with their original repetitive sequences. Finally, an two-stage amplification model, including amplification by intra-chain slide and mediated by hairpin-contained structure, was proposed to provide information for the study of nonspecific amplification of repetitive sequences and pathogenetic mechanisms of relevant diseases.