EMSA is not suitable for:
EMSA is excellent for qualitative and semi-quantitative evaluation of sequence-specific DNA-protein complex formation using short labeled probes of known sequence. Under non-denaturing conditions, binding affinity, specificity via competitor DNA and complex stoichiometry can be assessed. However, assay fundamentally depends on prior knowledge of DNA fragment sequence and cannot determine unknown nucleotide order. Determination of primary structure requires Sanger sequencing or next-generation sequencing technologies. Consequently, EMSA is unsuitable for DNA sequence determination, although it
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.