Accuracy and reproducibility of protein-DNA microarray technology
Accuracy and reproducibility of protein-DNA microarray technology
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DOI：
10.1007/10_2006_035
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发表时间：
2007-01-01
期刊：
影响因子：
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通讯作者：
Ragoussis, Jiannis
中科院分区：
文献类型：
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作者：
Field, Simon;Udalova, Irina;Ragoussis, Jiannis
由于需要在这一领域引入高通量技术，基于微阵列的方法用于理解蛋白质- dna相互作用在过去6年中得到了发展。蛋白质-DNA微阵列利用可以打印或合成大量DNA序列的芯片。任何dna结合蛋白都可以通过纯化样品或细胞/核萃取物进行检测，前提是有合适的检测系统。只需将蛋白质添加到微阵列载玻片表面，然后将其洗涤并与标记的分子进行至少一次进一步的孵育，该分子与感兴趣的蛋白质特异性结合。所获得的信号与每个DNA特征结合的DNA结合蛋白的水平成正比，从而可以计算出相对亲和力。可重复和准确定量蛋白质结合的关键因素是：微阵列表面化学；寡核苷酸长度；结合位点序列的位置；蛋白质和抗体的质量；还有杂交条件。
Microarray-based methods for understanding protein-DNA interactions have been developed in the last 6 years due to the need to introduce high-throughput technologies in this field. Protein-DNA microarrays utilise chips upon which a large number of DNA sequences may be printed or synthesised. Any DNA-binding protein may then be interrogated by applying either purified sample or cellular/nuclear extracts, subject to availability of a suitable detection system. Protein is simply added to the microarray slide surface, which is then washed and subjected to at least one further incubation with a labelled molecule which binds specifically to the protein of interest. The signal obtained is proportional to the level of DNA-binding protein bound to each DNA feature, enabling relative affinities to be calculated. Key factors for reproducible and accurate quantification of protein binding are: microarray surface chemistry; length of oligonucleotides; position of the binding site sequence; quality of the protein and antibodies; and hybridisation conditions.