Reproducible and inexpensive probe preparation for oligonucleotide arrays.
Reproducible and inexpensive probe preparation for oligonucleotide arrays.
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DOI：
10.1093/nar/29.13.e66
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发表时间：
2001-07-01
影响因子：
14.9
通讯作者：
Makrigiorgos, G M
中科院分区：
文献类型：
--
作者：
Zhang, Y;Price, B D;Makrigiorgos, G M
我们提出了一种制备用于微阵列杂交的核酸的新方法。DNA的定量和重复性是使用基于羟基的反应，该反应是由过氧化氢、铁(II)-EDTA和抗坏血酸引发的。在碎裂后，使用生物素化的补骨脂素类似物加上UVA光将核酸片段密集地生物素化，并在微阵列上杂交。这种非酶方法避免了与微阵列DNA制备相关的几个实际困难：缺乏与酶方法相关的可重现的片段模式；一些阵列设计需要大量标记的核酸，这通常与有限的起始材料相结合；以及与当前使用的生物素化方法相关的高成本。该方法适用于任何形式的核酸，但在将双链DNA应用于寡核苷酸阵列时尤其有用。通过将聚合酶链式反应产物与包被寡核苷酸的微球和聚合酶链式反应扩增的基因与Affymetrix癌症基因芯片微阵列杂交，验证了该方法的有效性。
We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.