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Sequencing RNA by a Combination of Exonuclease Digestion and Uridine Specific Chemical Cleavage Using MALDI-TOF

Overview
Journal Nucleic Acids Res
Publisher Oxford University Press
Specialty Biochemistry
Date 1998 Feb 28
PMID 9421498
Citations 12
Authors
D A Tolson
N H Nicholson
Affiliations
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Abstract

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.

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