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Do you know which modifications you off would block ligations?

 
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07/28/2025

Do you know which modifications you off would block ligations?

Normally oligo come with 5'OH and 3'OH, unless requested to have 5' Phosphate, ligation will not occur. 

Modification That Prevent Ligation

Modification Position Blocking Mechanism HPLC Compatibility Notes
3′ Phosphate (-P) 3′ end Blocks ligase activity ✓ Compatible with RP-HPLC Common in blocking unwanted elongation
3′ Inverted dT (3′-3′ linkage) 3′ end No 3′ OH; terminates strand ✓ Stable under HPLC Effective irreversible block
3′ Biotin / C3 Spacer 3′ end No hydroxyl; steric block ✓ Stable through HPLC Useful for streptavidin capture
3′ Amino Modifier (C6-NH2) 3′ end No 3′ OH; reactive handle ✓ Compatible with RP and IEX HPLC Used for downstream conjugation
3′ Hexanediol (Spacer 18) 3′ end Flexible hydrophilic blocker ✓ RP-HPLC compatible Also increases solubility
3′ Dideoxynucleotide (ddNTP) 3′ end Terminator; no 3′ OH ✓ HPLC stable Used in Sanger sequencing
No 5′ Phosphate 5′ end Ligation requires 5′-P ✓ Does not affect HPLC Use when 5′ ligation must be blocked
5′ Biotin / Amine / Dye 5′ end Steric hindrance; label blocks ligation ✓ Stable during HPLC Also enables detection or pull-down
2′-O-Methyl, 2′-F, LNA Internal or ends Conformational changes reduce ligation ✓ Stable in RP-HPLC Depends on sequence context
PEG Spacer (e.g., Spacer 9/18) Any position Bulky, flexible; impairs enzyme binding ✓ RP-HPLC compatible Spacer between moieties or to block activity