Sequencing RNA by a combination of exonuclease digestion and uridine specific chemical cleavage using MALDI-TOF

The determination of DNA sequences by partial exonuclease digestion followed by Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF) is a well established method. When the same procedure is applied to RNA, difficulties arise due to the small (1 Da) mass difference between th...

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Bibliographic Details
Published in:Nucleic acids research 1998-01, Vol.26 (2), p.446-451
Main Authors: Tolson, D. A., Nicholson, N. H.
Format: Article
Language:English
Subjects:
Aniline Compounds
Base Sequence
Endoribonucleases - metabolism
Exonucleases - metabolism
Hydrazines
Lasers
Mass Spectrometry - methods
RNA - chemistry
Sequence Analysis, RNA - methods
Uridine - chemistry
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Summary:The determination of DNA sequences by partial exonuclease digestion followed by Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF) is a well established method. When the same procedure is applied to RNA, difficulties arise due to the small (1 Da) mass difference between the nucleotides U and C, which makes unambiguous assignment difficult using a MALDI-TOF instrument. Here we report our experiences with sequence specific endonucleases and chemical methods followed by MALDI-TOF to resolve these sequence ambiguities. We have found chemical methods superior to endonucleases both in terms of correct specificity and extent of sequence coverage. This methodology can be used in combination with exonuclease digestion to rapidly assign RNA sequences.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/26.2.446