Differential display of genome subsets containing specific interspersed repeats

A genomic differential display method was developed that analyzes many restriction fragment length polymorphisms simultaneously. Interspersed repeat sequences were used to reduce DNA sample complexity and to target genomic subsets of interest. This work focused on trinucleotide repeats because of their importance in human inherited diseases. Immobilized repeat-containing oligonucleotides were used to capture genomic DNA fragments containing sequences complementary to the oligonucleotide. Captured fragments were amplified by PCR and fluorescently labeled using primers complementary to the repeat sequence and/or to the known sequences ligated to the ends of the restriction fragments. The labeled PCR fragments were displayed by size on a high-resolution automated fluorescent DNA sequencing instrument. Although there was a conservation in the overall pattern of displayed genome subsets, many clear and reproducible differences were detected when genomes from different individuals were compared. Fewer differences were detected within, than between, monozygotic twin pair genomes. In control experiments, the method distinguished between Huntington disease alleles with normal and expanded CAG repeat lengths.

本研究开发了一种可同时分析多类限制性片段长度多态性（restriction fragment length polymorphisms）的基因组差异显示（genomic differential display）技术。研究借助散布重复序列（interspersed repeat sequences）降低DNA样本的复杂度，并靶向目标基因组亚群。鉴于三核苷酸重复序列（trinucleotide repeats）在人类遗传性疾病中的重要作用，本研究聚焦于该类序列。实验中采用固定化的含重复序列寡核苷酸（oligonucleotide），捕获与该寡核苷酸互补的基因组DNA片段。捕获得到的片段经聚合酶链式反应（PCR）扩增后，使用与重复序列互补、或与连接至限制性片段（restriction fragment）末端的已知序列互补的引物（primer）进行荧光标记。标记后的PCR片段通过高分辨率自动化荧光DNA测序仪（high-resolution automated fluorescent DNA sequencing instrument）按分子量大小进行分离展示。尽管所展示的基因组亚群整体模式具有保守性，但对比不同个体的基因组时，可检测到大量清晰且可重复的差异。同卵双生子（monozygotic twin）基因组内部检测到的差异数量，显著少于双生子个体间的差异。在对照实验中，该方法可有效区分携带正常CAG重复长度与扩增CAG重复长度的亨廷顿病（Huntington disease）等位基因（allele）。