Which enzyme in SAGE is used to produce 12 bp tags?
In SAGE methodology, the critical step generating transcript-specific tags involves a tagging enzyme, a type IIS restriction endonuclease that binds recognition sequence but cleaves outside. Anchoring enzyme NlaIII initially cleaves complementary DNA. Linkers are ligated, then BsmFI, recognizing sequence within linker, cleaves 10 nucleotides away on one strand and 14 nucleotides on complementary strand, releasing a fragment containing linker plus approximately 10 nucleotides of transcript-derived sequence, processed to yield 12 base expression tags. Sequencing of concatenated tags enables digital counting of messenger RNAs without prior sequence information, providing transcriptome inventory.
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.