三核苷酸双链重复序列扩展合成特性及其机理

简单重复序列在各种生物基因组中广泛存在,同分子进化、遗传多样性、分子标记和某些遗传性疾病等密切相关．本文以全部32种18 bp三核苷酸双链重复序列作为研究对象,对它们在聚合酶作用下的扩展合成进行了系统研究．探讨了反应温度、序列本身等对扩展效率和产物长度的影响．结果显示,几乎所有的序列都能扩展变长．但序列对其扩展效率有很大影响：GC含量较多的短链,尤其是一条链中同时有G和C的短链扩展效率较高;全由AC和GT组成的双链也较易扩展．短链的最适扩展温度与短链GC组成呈一定的正相关关系．琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳分析发现,大部分产物单一性良好,且产物分子质量的大小随反应时间线性增加;随着反应温度升高,产物差异性增大．最后分析了双链重复序列的“复制滑移”扩展机理,有望为进一步研究重复序列的分子进化和基因检测中的非特异性扩增等奠定基础．

Expansion of Trinucleotide Repetitive Sequences and Its Mechanism

Simple repetitive sequences are widely present in genomes of most organisms. They are closely related to molecular evolution, genetic diversity, molecular marker and some hereditary diseases. In this article, all of the 18 bp trinucleotide repetitive double strand sequences were used, and their expansion by DNA polymerase was researched systematically. We discussed the factors including reaction temperature and sequence which influence the amplification efficiency and the length of product. The result showed that almost all of the repeats can be elongated, and the variance in sequence had great effect on the amplification efficiency. The repeats with more GC, especially those with G and C in one of the strands were much easier to grow. Duplexes with one strand containing only A and C were amplified a lot as well. The optimum reaction temperature and the GC content of the duplexes had a positive correlation. Agarose and polyacrylamide gel electrophoresis showed that the product has narrow size distribution of molecules, and the expansion has a linear dependence of the length of products on the reaction time. Differentiation of the products occurred more at higher reaction temperatures. At last, a slippage model demonstrating the mechanism of duplex expansion was discussed, and it is promising to be used for explanation of molecular evolution and unusual expansion of repetitive sequences during gene amplification and detection.