Rapid procedure for chemical sequencing of small oligonucleotides without ethanol precipitation.

We report here a procedure for the chemical sequencing of oligonucleotides that offers two advantages over the standard Maxam-Gilbert protocol for chemical sequencing (1). First, the precipitation of small DNAs, which is often highly variable and inefficient, is avoided, allowing quantitative sequence analysis or structure mapping of very small oligonucleotides. Second, the procedure is extremely rapid; samples can be loaded onto a sequencing gel within 90 minutes of starting the reactions. This procedure consists of three chemical reactions: the G reaction is the standard dimethyl sulfate (DMS) reaction, the G+A reaction is depurination with piperidinium formate (pH 2.0), and the T reaction is modification with potassium permanganate (KMnO4) (2). There is no C reaction, but the positions of C's can be inferred from the presence of background level cleavage in all three lanes. of and volumes to reduce salt artifacts during the buffer is not removed from the sample by precipitation, any buffer in the sample is ultimately onto reactions piperidinium