O'Connell C D, Atha D H, Jakupciak J P, Amos J A, Richie K l
DNA Technologies Group, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8311, USA.
Clin Genet. 2002 Jan;61(1):13-20. doi: 10.1034/j.1399-0004.2002.610103.x.
To provide the clinical diagnostics community with accurate protocols and measurements for the detection of genetic disorders, we have established a quantitative measurement program for trinucleotide repeats associated with human disease. In this study, we have focused on the triplet repeat associated with fragile X syndrome. Five cell lines obtained from the Coriell Cell Repository were analyzed after polymerase chain reaction (PCR) amplification and size separation. These cell lines were reported to contain CGG repeat elements (ranging from 29 to 110 repeats). Our initial measurements focused on measurement variability: (a) between slab-PAGE and capillary (CE) separation systems (b) interlane variability (slab-PAGE) (c) intergel variability, and (d) variability associated with amplification. Samples were run in triplicate for all measurements, and the analysis performed using Gene Scan analysis software. The repeat sizes were verified by DNA sequence analyzes. The standard deviations for interlane measurements in slab-gels ranged from 0.05 to 0.35. There was also little variation in size measurements performed on different gels and among PCR amplifications. The CGG repeat measurements performed by capillary electrophoresis were more precise, with standard deviations ranging from 0.02 to 0.29. The slab-PAGE and CE size measurements were in agreement except for the pre-mutation alleles, which yielded significantly smaller sizes by CE.
为了向临床诊断领域提供用于检测遗传疾病的准确方案和测量方法，我们建立了一个针对与人类疾病相关的三核苷酸重复序列的定量测量程序。在本研究中，我们重点关注与脆性X综合征相关的三联体重复序列。从科里尔细胞库获得的5种细胞系在经过聚合酶链反应（PCR）扩增和大小分离后进行了分析。据报道，这些细胞系含有CGG重复元件（重复次数从29次到110次不等）。我们最初的测量集中在测量变异性上：（a）平板聚丙烯酰胺凝胶电泳（slab-PAGE）和毛细管电泳（CE）分离系统之间；（b）平板聚丙烯酰胺凝胶电泳的泳道间变异性；（c）凝胶间变异性；以及（d）与扩增相关的变异性。所有测量均对样品进行三次重复运行，并使用基因扫描分析软件进行分析。重复序列大小通过DNA序列分析进行验证。平板凝胶中泳道间测量的标准偏差范围为0.05至0.35。在不同凝胶上以及PCR扩增之间进行的大小测量也几乎没有差异。通过毛细管电泳进行的CGG重复序列测量更为精确，标准偏差范围为0.02至0.29。除了前突变等位基因外，平板聚丙烯酰胺凝胶电泳和毛细管电泳的大小测量结果一致，前突变等位基因通过毛细管电泳测得的大小明显更小。
