Accuracy and reproducibility of protein-DNA microarray technology.
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.
Field
Simon
S
Wellcome Trust Centre for Human Genetics, University of Oxford, 7 Roosevelt Drive, Oxford OX3 7BN, UK.
Udalova
Irina
I
Ragoussis
Jiannis
J
D001665:Binding Sites; D001709:Biotechnology / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA