Why is formaldehyde used in denaturing electrophoresis buffers?
RNA readily forms stable secondary structures such as hairpins and stem-loops via Watson-Crick pairing, altering electrophoretic mobility and preventing accurate size estimation. Denaturing electrophoresis requires agents that disrupt hydrogen bonding. Formaldehyde at 1 to 2.2 M reacts with imino groups of adenine, guanine, and cytosine, preventing base pairing and maintaining RNA as linear molecules. Glyoxal and formamide act similarly. Formaldehyde does not stain, fix DNA, or remove proteins as primary purpose; its function is structural disruption for accurate RNA sizing.
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.