Analysis of enzymatic DNA sequencing reactions by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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.
Shaler
T A
TA
Molecular Physics Laboratory, SRI International, Menlo Park, CA 94025, USA.
Tan
Y
Y
Wickham
J N
JN
Wu
K J
KJ
Becker
C H
CH
D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000032:analysis / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D007834:Lasers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic; D014462:Ultrafiltration
D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase