Hydrolysis of nucleoside residues in DNA occurs to generate abasic sites. Most commonly, dA sites are hydrolyzed causing depurination and leading to abasic residues. For researchers trying to determine if their source of depurination in chemical synthesis of DNA is reagent, fluidics or protocol-based, we offer a depurination-resistant dA monomer. A new chemical method allows the generation of abasic sites in double and single stranded oligonucleotides using very mild specific conditions and with very low probability of side reactions. The original Abasic Phosphoramidite (10-1924) has been discontinued since it exhibits low coupling efficiency and the post-synthesis chemistry is fairly challenging. Abasic II Phosphoramidite1 is the replacement for the preparation of a true abasic site. This product has the advantage of simplicity in that the silyl group is removed post-synthesis using aqueous acetic acid. dSpacer has also been used successfully as a mimic of the highly base-labile abasic site.
Details
Usage
Coupling: 6 minute coupling time recommended.
Deprotection: Synthesize using acetyl-protected dC and deprotect in 30% Ammonium Hydroxide/40% Methylamine 1:1 (AMA) at 65°C for 10 min OR Synthesize using dmf-dG and deprotect in EtOH/30% Ammonium Hydroxide (1:3) for 24 hrs at room temperature. To generate the abasic site, dry the oligo down and take up in 0.25mL 80% Acetic acid. After 30 min at room temperature, add 0.25mL water and let sit for an additional 4 hrs. Quench with 0.5mL 2M TEAA and desalt on a Glen Gel-Pak™ column or equivalent. Store the oligo at pH 6-7 at 4°C or frozen. Do not dry; drying results in cleavage of the abasic site. See Technical Bulletin for details (http://www.glenresearch.com/Technical/TB_Abasic II-1.pdf).
Specifications
Diluent
Anhydrous Acetonitrile
Storage
Freezer storage, -10 to -30°C, dry
Stability
2-3 days
Dilution/Coupling Data
The table below show pack size data and, for solutions, dilution and approximate coupling based on normal priming procedures.
