Sequencing oligonucleotides with blocked termini using exonuclease digestion and electrospray mass spectrometry.
A method for sequencing ODNs with both termini blocked using mass spectrometry (MS) is reported. The ladder sequencing method is based on our investigation and understanding of critical factors affecting snake venom phosphodiesterase (SVP) digestion of such ODNs. To produce sequence ladders suitable for MS analysis, digestion conditions such as SVPs from three snake species and pH values of digestion buffer were investigated. SVP of Crotalus duressus terrificus (SVP I) was found to be the most suitable for sequencing ODNs 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 sequencing 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 digestion buffer of desired pH values, sequence ladders which are recorded as MWs of truncated ODNs from SVP digestion are obtained. Examples of sequencing ODNs up to 26 bases long with both termini blocked are demonstrated in this work.
Wu
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Abbott Laboratories, Diagnostics Division, 100 Abbott Park Road, Abbott Park, Illinois 60064, USA. Huaiqin.Wu@abbott.com
Aboleneen
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D000818:Animals; D001483:Base Sequence; D001639:Bicarbonates / Q000494:pharmacology; D002021:Buffers; D005092:Exonucleases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D017422:Sequence Analysis, DNA / Q000379:methods*; D012910:Snake Venoms / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001639:Bicarbonates; D002021:Buffers; D009841:Oligonucleotides; D012910:Snake Venoms; C027043:ammonium bicarbonate; D005092:Exonucleases; D010727:Phosphoric Diester Hydrolases