Solid-state DNA sizing by atomic force microscopy.
Atomic force microscopy (AFM) allows rapid, accurate, and reproducible visualization of DNA adsorbed onto solid supports. The images reflect the lengths of the DNA molecules in the sample. Here we propose a solid-state DNA sizing (SSDS) method based on AFM as an analytical method for high-throughput applications such as finger-printing, restriction mapping, +/- screening, and genotyping. For this process, the sample is first deposited onto a solid support by adsorption from solution. It is then dried and imaged under ambient conditions by AFM. The resulting images are subjected to automated determination of the lengths of the DNA molecules on the surface. The result is a histogram of sizes that is similar to densitometric scans of DNA samples separated on gels. A direct comparison of SSDS with agarose gel electrophoresis for +/- screening shows that it produces equivalent results. Advantages of SSDS include reduced sample size (i.e., lower reagent costs), rapid analysis of single samples, and potential for full automation using available technology. The high sensitivity of the method also allows the number of polymerase chain reaction cycles to be reduced to 15 or less. Because the high signal-to-noise ratio of the AFM allows for direct visualization of DNA-binding proteins, different DNA conformations, restriction enzymes, and other DNA modifications, there is potential for dramatically improving the information content in this type of analysis.
Fang
Y
Y
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Spisz
T S
TS
Wiltshire
T
T
D'Costa
N P
NP
Bankman
I N
IN
Reeves
R H
RH
Hoh
J H
JH
D000327:Adsorption; D001331:Automation; D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000648:ultrastructure; D004587:Electrophoresis, Agar Gel; D007091:Image Processing, Computer-Assisted / Q000379:methods; D018625:Microscopy, Atomic Force / Q000379:methods*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012996:Solutions
D012996:Solutions; D004247:DNA