Key featuresShow Hide
- Useful for mutagenic studies involving methylation.
- Acetyl protection to avoid branching when using DCI as an activator
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Methylating agents are common carcinogens which function by methylation of nucleobases in DNA. Cellular polynucleotides are alkylated by endogenous components, such as S-adenosylmethionine. Chemically, SN1 agents such as alkylnitrosourea and N-alkyl-N-nitro-N-nitrosoguanidine will react with the N7 position of guanine, N3 of adenine, O6 of guanine, O2 or O4 of pyrimidines, and the non-phosphodiester oxygen atoms of the phosphate backbone. In contrast, SN2 chemical agents such as methyl methanesulfonate and dimethyl sulfate react primarily with the N1 position of adenine (1-Methyl-2'-deoxyadenosine) and N3 of cytosine. To examine some of these resulting mutagenic effects, the methylated products O6-Me-dG- CE Phosphoramidite, N6-Me-dA-CE Phosphoramidite, and O4-Me-dT-CE Phosphoramidite can be incorporated in oligonucleotides. To avoid chain branching during synthesis when using DCI as activator, N6-Me-dA is offered with acetyl protection. O6-Me-dG- CE Phosphoramidite has classic iBu protection.
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
No changes are required from the standard method recommended by the synthesiser manufacturer. Coupling is as per standard nucleoside amidites.
Cleavage & Deprotection
LK2018 and LK2025 -
- Empty the synthesis resin from the column into a sample tube.
- Add 900μl anhydrous MeOH and 100μl DBU (1,8-Diazabicyclo [5.4.0]undec-7-ene) to the resin in the sample tube and cap tightly.
- Place the tube in a dry dark place and leave it for 5 days.
- Reduce to small volume.
- Add 1ml of 10mM aqueous sodium hydroxide solution and desalt or purify the oligonucleotide using standard procedures.
LK2019 - Use AMA at room temperature for 2h. Employ dmf-G and Ac-C within the sequence.
Storage & Stability
All phosphoramidites are stored refrigerated at a maximum of 2-8°C. Stability in solution is 2-3 days.