Improved oligonucleotide sequencing by alkaline phosphatase and exonuclease digestions with mass spectrometry.
The combination of exonuclease digestion and mass spectrometry has been widely used for sequencing oligonucleotides. During an exonuclease digestion, rapid buildup in the concentration of nucleotides produces strong signal of nucleotide cluster ions in electrospray ionization-mass spectrometry, especially for oligonucleotides with greater than 25 bases. This leads to poor signal/noise ratio in the reconstructed molecular weight spectra of late digestion products due to artifact peaks from nucleotide cluster ions. Here we report a procedure that eliminates the effect of the cluster ions. In this method, alkaline phosphatase is added with snake venom phosphodiesterase to the oligonucleotide solution to convert the interfering nucleotides into noninterfering nucleosides, and the collision-induced dissociation spectrum of the dimeric oligonucleotide at the end of the digestion is obtained to determine the sequence of the last two bases at the 5'-terminus of the oligonucleotide. With this approach, the signal/noise ratio of the reconstructed molecular weight spectrum is greatly improved for relatively large oligonucleotides, and only a single digestion is needed for sequencing.
Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064, USA. Huaquin.Wu@abbott.com
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D000469:Alkaline Phosphatase / Q000737:chemistry*; D005092:Exonucleases / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D009841:Oligonucleotides / Q000032:analysis*; D017422:Sequence Analysis, DNA
D009841:Oligonucleotides; D005092:Exonucleases; D000469:Alkaline Phosphatase