Dimethyl sulfate can affect the base-specific cleavage of DNA by attacking the imidazole ring present in guanine. [15] Dimethyl sulfate also methylates adenines in single-stranded portions of DNA (for example, those containing proteins such as RNA polymerase that gradually melt and re-anneal DNA). After reannealing, these methyl groups interfere with adenine-guanine base pairing. The DNA can then be cleaved at single-stranded regions (anywhere with methylated adenines) using nuclease S1. This is an important technique for analyzing protein-DNA interactions.

Although dimethyl sulfate is very effective and affordable, its toxicity has prompted the use of other methylating agents. Methyl iodide is a reagent used for O-methylation, like dimethyl sulfate, but is less harmful and more expensive. [13] Dimethyl carbonate is less reactive and much less toxic than dimethyl sulfate and methyl iodide. [16] High pressure can be used to accelerate the methylation of dimethyl carbonate. In general, the toxicity of methylating reagents correlates with their efficiency as transmethylating reagents.

Dimethyl sulfate is carcinogenic [10] and mutagenic, highly toxic, corrosive, and environmentally hazardous. [17] It is absorbed through the skin, mucous membranes, and gastrointestinal tract and can cause fatal delayed respiratory reactions. Eye reactions are also common. There is no strong odor or immediate irritation to warn of lethal concentrations in the air. LD50 (acute, oral) is 205 mg/kg (rat) and 140 mg/kg (mice), LC50 (acute) is 45 ppm / 4 hours (rat). [18] The vapor pressure of 65 Pa[19] is large enough to produce lethal concentrations in air by evaporation at 20 °C. Delayed toxicity allows potentially fatal exposure to occur before any warning symptoms appear. [17] Symptoms may be delayed for 6-24 hours. Concentrated solutions of bases (ammonia, alkali) can be used to hydrolyze small spills and residues on contaminated equipment, but the reaction may become violent with the addition of dimethyl sulfate (see Chemical Safety Card). Although the compound is hydrolyzed, it cannot be assumed that treatment with water will decontaminate it.