Analysis of enzymatic DNA sequencing reactions by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
| Autor: | Yuping Tan, Kuang Jen Wu, Juanita N. Wickham, Thomas A. Shaler, Christopher H. Becker |
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| Rok vydání: | 1995 |
| Předmět: |
Detection limit
Chromatography Base Sequence Sequence analysis Chemistry Lasers Molecular Sequence Data Organic Chemistry Analytical chemistry DNA Single-Stranded Ultrafiltration T7 DNA polymerase DNA-Directed DNA Polymerase Sequence Analysis DNA Templates Genetic Mass spectrometry Mass Spectrometry DNA sequencing Analytical Chemistry Surface-enhanced laser desorption/ionization chemistry.chemical_compound Primer (molecular biology) Spectroscopy DNA |
| Zdroj: | Rapid Communications in Mass Spectrometry. 9:942-947 |
| ISSN: | 1097-0231 0951-4198 |
| DOI: | 10.1002/rcm.1290091015 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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