Streamlined assessment of gene variants by high resolution melt profiling utilizing the ornithine transcarbamylase gene as a model system.
Streamlined assessment of gene variants by high resolution melt profiling utilizing the ornithine transcarbamylase gene as a model system.
复制标题
利用鸟氨酸转氨甲酰酶基因作为模型系统，通过高分辨率熔解分析简化基因变异的评估。
DOI：
10.1002/humu.20558
复制
发表时间：
2007
期刊：
影响因子：
3.9
通讯作者：
Tuchman,Mendel
中科院分区：
文献类型：
--
作者：
Dobrowolski,StevenF;Ellingson,ClintonE;Caldovic,Ljubica;Tuchman,Mendel
鸟氨酸转氨甲酰酶（OTC）缺乏症是一种X连锁的半显性遗传性疾病，也是最常见的尿素循环遗传缺陷。OTC基因的分子遗传学检测对于临床诊断、携带者检测和产前诊断至关重要。在整个OTC基因中观察到私人突变，报告了超过340种致病突变。采用高分辨率解链分析，以96孔板形式对10个编码区及其内含子侧翼进行同质分析。在PCR运行中扩增范围为146 bp至266 bp的10个DNA片段。一个共同的分析条件，同时产生所有10个片段的熔解曲线。为了简化分析，在选定的全外显子检测和单独的多重基因分型检测中，使用熔解曲线对照进行冗余评估，对常见多态性变体产生的异常曲线进行分类。通过从熔解曲线分析板回收染料染色的扩增产物作为DNA测序模板，进一步简化了检测。本文描述了23例OTC缺陷患者的OTC基因的综合分析。该系统提供了一种快速定位序列变体的方法，显著减少了对DNA测序的需求，并且适用于其他基因和疾病。Hum Mutat 28（11），1133-1140，2007年。2007年出版Wiley利斯公司
Ornithine transcarbamylase (OTC) deficiency is an X‐linked, semidominant genetic disorder and the most prevalent inherited defect of the urea cycle. Molecular genetic testing of the OTC gene is critically important for clinical diagnosis, carrier testing, and prenatal diagnosis. Private mutations are observed throughout the OTC gene with more than 340 reported disease‐causing mutations. High‐resolution melt profiling was adapted to perform homogeneous analysis of the 10 coding regions and their intronic flanks in a 96‐well plate format. The 10 DNA fragments ranging from 146 bp to 266 bp are amplified in a PCR run. A common analysis condition simultaneously generates melting profiles from all 10 fragments. To streamline analysis, deviant profiles resulting from common polymorphic variants are triaged using redundant assessment with melt profile controls in selected whole‐exon assays and a separate multiplex genotyping assay. The test is further streamlined by recovering dye‐stained amplification product from the melt profiling plate to serve as DNA sequencing template. Described herein is the comprehensive analysis of the OTC gene in 23 OTC‐deficient patients. This system provides a rapid means to localize sequence variants, markedly reducing the need for DNA sequencing, and is applicable to other genes and disorders. Hum Mutat 28(11), 1133–1140, 2007. Published 2007 Wiley‐Liss, Inc.