Sequencing oligonucleotides by exonuclease digestion and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
A protocol was developed for sequencing oligonucleotides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Oligonucleotides were partially hydrolyzed in separate time-course digestions with 3' --> 5' and 5' --> 3' acting phosphodiesterases in a MALDI-compatible buffer or in the MALDI matrix itself. The partial digests were analyzed by MALDI-TOF mass spectrometry employing an instrument equipped with delayed ion extraction and the sequence was inferred from the mass differences between adjacent peaks. Resolution, mass accuracy, and sensitivity were considerably enhanced with delayed extraction, in comparison with the standard MALDI technique. Much longer lengths of DNA can be unambiguously sequenced with delayed extraction MALDI compared with standard MALDI-MS.
Smirnov
I P
IP
PerSeptive Biosystems, 500 Old Connecticut Path, Framingham, Massachusetts, 01701, USA.
Roskey
M T
MT
Juhasz
P
P
Takach
E J
EJ
Martin
S A
SA
Haff
L A
LA
D001483:Base Sequence; D005092:Exonucleases; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors
D009841:Oligonucleotides; D005092:Exonucleases