Enhanced high density oligonucleotide array-based sequence analysis using modified nucleoside triphosphates
Enhanced high density oligonucleotide array-based sequence analysis using modified nucleoside triphosphates
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DOI：
10.1093/nar/26.21.4975
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发表时间：
1998-11-01
影响因子：
14.9
通讯作者：
Collins, FS
中科院分区：
文献类型：
--
作者：
Hacia, JG;Woski, SA;Collins, FS
设计了由>96 000个寡核苷酸组成的高密度寡核苷酸阵列（DNA芯片）对，以筛选遗传性乳腺癌和卵巢癌BRCA 1基因的整个5.53 kb编码区，以确定纯合和杂合状态下所有可能的序列变化。通过使用含有T3和T7 RNA聚合酶启动子尾的引物对单个BRCA 1外显子进行PCR扩增，然后进行体外转录和部分片段化反应，生成单链RNA靶标。来自含有四种天然碱基的靶点与芯片上>5592种不同的完全互补25 mer寡核苷酸探针的荧光杂交信号变化超过两个数量级。为了检查rU.dA和rA.dT靶标探针碱基对这种变异性的热力学贡献，将修饰的尿苷[5-甲基尿苷和5-（1-丙炔基）-尿苷]和修饰的腺苷（2，6-二氨基嘌呤核苷）5 '-三磷酸掺入BRCA 1靶标中。基于来自相对于靶序列含有中心定位的单碱基对错配的>33 200个探针的杂交信号来评估杂交特异性。含有5-甲基尿苷的目标显示出有希望的局部增强杂交信号，特别是在富含嘧啶的靶区，同时保持单核苷酸错配杂交特异性与未修饰的目标。
Pairs of high density oligonucleotide arrays (DNA chips) consisting of >96 000 oligonucleotides were designed to screen the entire 5.53 kb coding region of the hereditary breast and ovarian cancer BRCA1 gene for all possible sequence changes in the homozygous and heterozygous states. Single-stranded RNA targets were generated by PCR amplification of individual BRCA1 exons using primers containing T3 and T7 RNA polymerase promoter tails followed by in vitro transcription and partial fragmentation reactions. Fluorescent hybridization signals from targets containing the four natural bases to >5592 different fully complementary 25mer oligonucleotide probes an the chip varied over two orders of magnitude. To examine the thermodynamic contribution of rU.dA and rA.dT target.probe base pairs to this variability, modified uridine [5-methyluridine and 5-(1-propynyl)-uridine)] and modified adenosine (2,6-diaminopurine riboside) 5'-triphosphates were incorporated into BRCA1 targets. Hybridization specificity was assessed based upon hybridization signals from >33 200 probes containing centrally localized single base pair mismatches relative to target sequence. Targets containing 5-methyluridine displayed promising localized enhancements in hybridization signal, especially in pyrimidine-rich target tracts, while maintaining single nucleotide mismatch hybridization specificities comparable with those of unmodified targets.