Petronis A, Heng H H, Tatuch Y, Shi X M, Klempan T A, Tsui L C, Ashizawa T, Surh L C, Holden J J, Kennedy J L
Neurogenetics Section, Hospital for Sick Children, Toronto.
Am J Med Genet. 1996 Feb 16;67(1):85-91. doi: 10.1002/(SICI)1096-8628(19960216)67:1<85::AID-AJMG15>3.0.CO;2-L.
Recently, unstable trinucleotide repeats have been shown to be the etiologic factor in seven neuropsychiatric diseases, and they may play a similar role in other genetic disorders which exhibit genetic anticipation. We have tested one polymerase chain reaction (PCR)-based and two hybridization-based methods for direct detection of unstable DNA expansion in genomic DNA. This technique employs a single primer (asymmetric) PCR using total genomic DNA as a template to efficiently screen for the presence of large trinucleotide repeat expansions. High-stringency Southern blot hybridization with a PCR-generated trinucleotide repeat probe allowed detection of the DNA fragment containing the expansion. Analysis of myotonic dystrophy patients containing different degrees of (CTG)n expansion demonstrated the identification of the site of trinucleotide instability in some affected individuals without any prior information regarding genetic map location. The same probe was used for fluorescent in situ hybridization and several regions of (CTG)n/(CAG)n repeats in the human genome were detected, including the myotonic dystrophy locus on chromosome 19q. Although limited at present to large trinucleotide repeat expansions, these strategies can be applied to directly clone genes involved in disorders caused by large expansions of unstable DNA.
最近，不稳定的三核苷酸重复序列已被证明是七种神经精神疾病的病因，并且它们可能在其他表现出遗传早现的遗传疾病中发挥类似作用。我们测试了一种基于聚合酶链反应（PCR）的方法和两种基于杂交的方法，用于直接检测基因组DNA中不稳定的DNA扩增。该技术采用以总基因组DNA为模板的单引物（不对称）PCR，以有效筛选大的三核苷酸重复扩增的存在。用PCR产生的三核苷酸重复探针进行高严谨度的Southern印迹杂交，可检测到含有扩增的DNA片段。对含有不同程度（CTG）n扩增的强直性肌营养不良患者的分析表明，在一些受影响个体中可鉴定出三核苷酸不稳定位点，而无需任何有关遗传图谱位置的先验信息。相同的探针用于荧光原位杂交，并检测到人类基因组中（CTG）n/（CAG）n重复的几个区域，包括19号染色体上的强直性肌营养不良基因座。尽管目前仅限于大的三核苷酸重复扩增，但这些策略可用于直接克隆涉及由不稳定DNA的大扩增引起的疾病的基因。
