Sequencing oligonucleotides with blocked termini using exonuclease digestion and electrospray mass spectrometry

A method for sequencing ODNs with both termini blocked using mass spectrome try (MS) is reported. The ladder sequencing method is based on our investig ation and understanding of critical factors affecting snake venom phosphodi esterase (SVP) digestion of such ODNs. To produce sequence ladders suitable for MS analysis, digestion conditions such as SVPs from three snake specie s and pH values of digestion buffer were investigated. SVP of Crotalus dure ssus terrificus (SVP I) was found to be the most suitable for sequencing OD Ns with both termini blocked. The pH value of 9.4, which is optimal for SVP digestion of unmodified ODNs, was found to be unsuitable for ladder sequen cing ODNs with both termini blocked. Instead, digestion in a wide range of pH values (pH 5-8), including rarely used acidic conditions,was found to be necessary to obtain otherwise unobtainable sequence information. With dige stion buffer of desired pH values, sequence ladders which are recorded as M Ws of truncated ODNs from SVP digestion ar-e obtained. Examples of sequenci ng ODNs up to 26 bases long with both termini blocked are demonstrated in t his work. (C) 2000 Academic Press.