A system for on-line detection and resolution of radiolabeled DNA molecules and its application to automated DNA sequence analysis.
We describe a system for the real-time detection of radioactively labeled DNA molecules in gel matrix, and we demonstrate the application of this system to DNA sequence analysis. DNA sequencing reactions prepared by the Sanger chain termination method are resolved by electrophoresis on 8% polyacrylamide gels. During electrophoresis the 32P-labeled DNA fragments are detected by solid state detectors positioned 22 cm from the top surface of the gel. This system is able to resolve a DNA sequence of 300 bases or greater. Optimized protocols that allow sequence information to be obtained from single stranded and double-stranded templates are described. A linear relationship exists between the input dpm and the integrated peak values over a 20-fold range indicating that accurate DNA quantitation is also possible using this system.
Toneguzzo
F
F
EG&G Biomolecular, Natick, MA 01760.
Beck
J
J
Cahill
P
P
Ciarkowski
M
M
Page
G
G
Glynn
S
S
Hungerman
E
E
Levi
E
E
Ikeda
R
R
McKenney
K
K
D001331:Automation; D001483:Base Sequence; D001610:Beta Particles; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D003627:Data Interpretation, Statistical; D004591:Electrophoresis, Polyacrylamide Gel; D004867:Equipment Design; D005821:Genetic Techniques / Q000295:instrumentation*; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D010761:Phosphorus Radioisotopes; D004247:DNA