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Design and Validation of a Conformation-Sens itive Capillary Electrophoresis System f or MutationIdentification of the COL7A1 Gene with Automated Peak Comparison

机译:Conformation-Sens敏感的设计和验证f或毛细管电泳系统MutationIdentification COL7A1基因自动化的峰值比较

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摘要

Dystrophic epidermolysis bullosa is a heritable skin disease in which blisters occur because of a defect in type VII collagen resulting from mutations in the COL7A1 gene that is composed of 118 exons. Although a few mutations are specific to certain populations owing to founder effects, and although a few mutational hotspots exist, most mutations are unique to families and can be found scattered throughout the entire COL7A1 gene. This emphasizes the need for a sensitive, reliable, and efficient mutation scanning technique. Therefore, we developed a conformation-sensitive capillary electrophoresis (CSCE) system for COL7A1 mutation scanning. Here we report on the design and validation of this system. The CSCE technique is based on the principle of heteroduplex formation when polymerase chain reaction—amplified DNA fragments containing heterozygous sequence changes are slowly reannealed. These fluorescently labeled fragments have different migration characteristics and can be detected on a multi-capillary automated sequencer. Validation was performed by analysis of 29 known COL7A1 sequence changes, covering 33% of amplicons. After optimization of the conditions, all 29 sequence changes were detected by the CSCE system, irrespective of length or CG-content of amplicons and position of sequence changes, reflecting an analytical sensitivity of 90.2-100% (95% confidence interval). We conclude that this CSCE system is a rapid, reliable, cost-effective, and highly sensitive way of mutation scanning for COL7A1 in a molecular genetics service laboratory.
机译:营养不良的表皮松解大疱是可遗传的皮肤病的水泡的发生第七型胶原蛋白产生的缺陷由COL7A1基因的突变118个外显子。对特定人群由于创始人的影响,尽管一些突变热点存在,大多数突变是独特的家庭,可以发现分散在整个COL7A1基因。可靠和高效突变扫描技术。conformation-sensitive毛细管电泳(中国建筑)COL7A1基因突变扫描系统。我们的设计和验证报告系统。heteroduplex形成时的原则聚合酶链reaction-amplified DNA片段含有杂合的变化序列慢慢再退火。碎片有不同的迁移和能被探测到的特征multi-capillary自动测序仪。是由分析已知COL7A1 29序列变化,覆盖33%的扩增子。优化后的条件,所有29由中国建筑序列变化检测系统,无论长度或cg含量序列的扩增子和位置的变化,反映出一个分析灵敏度为90.2 -100%(95%置信区间)。中国建筑体系是一种快速、可靠、符合成本效益,和高度敏感的突变扫描方式在分子遗传学COL7A1服务实验室。

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