Analysis of enzymatic DNA sequencing reactions by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry
- 1 January 1995
- journal article
- research article
- Published by Wiley in Rapid Communications in Mass Spectrometry
- Vol. 9 (10), 942-947
- https://doi.org/10.1002/rcm.1290091015
Abstract
The products from base-specific, dideoxy-nucleotide chain-termination DNA sequencing reactions catalyzed by the modified T7 DNA polymerase have been analyzed by using the technique of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Preliminary experiments were performed to determine detection limits for a synthetic mixture of mixed-base single-stranded DNA which contained a 14-mer, a 21-mer, and a 41-mer; acceptable spectra, showing peaks for each component, were obtainable for samples that contained as little as 5 fmol per component. Initial sequencing reactions were therefore carried out on 2-pmol amounts of a short synthetic template that was 45 nucleotides in length, employing 2 pmol of 12-mer as the primer strand. This provided readable sequence information out to the 19th base past the primer. Using a 21-mer primer, nearly the entire sequence of the template could be read.Keywords
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