What is the role of EcoP151 in CAGE?
After cap-trapping and cDNA synthesis, CAGE adaptors containing the recognition motif for EcoP151, a type III restriction enzyme, are ligated upstream of the cDNA. EcoP151 cuts DNA at a fixed distance of 25 to 27 base pairs downstream of its site, independent of sequence context. This property generates uniform CAGE tags of approximately 27 nucleotides originating from the very 5′ end of transcripts. The length is optimal for unique genomic alignment while preserving TSS information. It does not label mRNA or ligate probes directly; its role is strictly tag definition.
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.