High-throughput single-molecule DNA screening based on electrophoresis.
In electrophoresis, the migration velocity is used for sizing DNA and proteins or for distinguishing molecules based on charge and hydrodynamic radius. Many protein and DNA assays relevant to disease diagnosis are based on such separations. However, standard protocols are not only slow (minutes to hours) but also insensitive (many molecules in a detectable band). We successfully demonstrated a high-throughput imaging approach that allows determination of the individual electrophoretic mobilities of many molecules at a time. Each measurement only requires a few milliseconds to complete. This opens up the possibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification. The purpose is not to separate the DNA molecules but to identify each one on the basis of the measured electrophoretic mobility. We developed three different procedures to measure the individual molecular mobilities. The results correlate well with capillary electrophoresis (CE) experiments for the same samples (2-49 kb dsDNA) under identical separation conditions. The implication is that any electrophoresis protocols from slab gels to CE should be adaptable to single-molecule screening for disease diagnosis.
Shortreed
M R
MR
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, 50011, USA.
Li
H
H
Huang
W H
WH
Yeung
E S
ES
D002021:Buffers; D004396:Coloring Agents; D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary
D002021:Buffers; D004396:Coloring Agents; D004247:DNA