Strategies for mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays
Strategies for mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays
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DOI：
10.1101/gr.8.12.1245
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发表时间：
1998-12-01
期刊：
影响因子：
7
通讯作者：
Collins, FS
中科院分区：
文献类型：
--
作者：
Hacia, JC;Sun, B;Collins, FS
具有复杂基因组结构的大基因的突变分析在医学遗传学中起重要作用。与当前突变筛选方案相关的技术限制已越来越重视开发新技术，以简化这些程序。高密度阵列> 90,000-寡核苷酸探针的长度为25个核苷酸，旨在筛选ARI基因9.17-kb编码区域中所有可能的杂合种系突变。在基于DNA芯片的杂交分析中快速开发多exon PCR扩增方案的策略是在准备62个ATM编码外显子的目标中的。改进了解释来自两色实验的数据的算法，其中开发了参考和测试样品与阵列共同杂交。在一项盲目的研究中，准确地检测到了22个基因组DNA样品中的18个不同的杂合子中的17个，其中8个不同的杂合子和8个不同的纯合序列变体与5个假阳性呼叫进行了准确检测到> 200 kb。在多个样本中发现的八个杂合序列变化中，在所有情况下都检测到六个。检测到其他突变扫描方法在这些相同样品上未发现的五种未报告的序列变化，这些序列变化未发现，从而导致ATM蛋白的氨基酸变化或过早截断。基于DNA芯片的测定应在复杂基因的高通量序列分析中起宝贵的作用。
Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were designed to screen For all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ARI gene. A strategy For rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms For interpreting data From two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.