Table 2 Advantages and disadvantages of techniques that study DNA-binding sites
From: DNA–protein interaction studies: a historical and comparative analysis
Technique [References] | Technique | Applications | Case studies | |
|---|---|---|---|---|
Pros | Cons | |||
In vivo methods consider factors existing in cells and enable the analysis of the transcription of particular alleles useful to study the interactions of a long nucleic acid with several binding proteins Signal is observed in binding equilibrium conditions | Low throughput “Protective” footprinting usually presents a background In vitro methods disregard factors existing in cells Need improvement of binding by the protein under study and the nucleic acid modification reaction The detection is not too sensitive The results obtained may not be so distinct | Discover the precise sequence of the nucleic acid that binds to a protein “Interference” footprinting: investigate the impact of each nucleotide on the binding affinity | Manosas et al. [55] | |
Base analogues [3] | Provides accurate results Access the relative affinity of a protein for DNA fragments that lack particular groups on a base | Expensive and long procedure | Identify the contribution of each base to the DNA-binding affinity of a protein Understand the contribution of crucial interactions to the binding | |
Provides accurate results Does not depend on cloning Does not require the analysis of phenotypes SELEX-SAGE provides binding-site models with a higher accuracy High-throughput SELEX provides better fits to the selected site distributions | Becomes more difficult when dealing with TFs that bind to several genes | Rapid selection of the oligonucleotides that have appropriate binding affinity to a molecular target from a library of randomly generated oligonucleotides | Prabu et al. [45] | |