ABSTRACT
Single cell analysis circumvents the need to average data from large populations by observing each cell individually, thus enabling the analysis of cell-to-cell variability. The ability to work on this scale presents many new opportunities for the life sciences and biomedical applications. Microfluidics has become a tool of choice for such studies and electrowetting on dielectric (EWOD) technology is well adapted for samples with reduced size and biological studies at the single cell level. In the present manuscript, for the first time, we present an integrated and automated system based on EWOD that can process the complete workflow on a single device, from the isolation of a single cell to mRNA purification and gene expression analysis.
Subject(s)
Cell Separation/instrumentation , Electrowetting/instrumentation , Microfluidic Analytical Techniques/instrumentation , Multiplex Polymerase Chain Reaction/instrumentation , RNA, Messenger/analysis , RNA, Messenger/isolation & purification , Cell Line , Equipment Design , HumansABSTRACT
The rate of cyclization of lambda DNA varies over more than 6 orders of magnitude, from 3.2 x 10(-7) s-1 to 2 s-1, in a Tris-EDTA buffer as a function of spermidine concentration. This variation is strictly correlated with the conformation of the chain. The highest rates are obtained when the chain is collapsed into a dense globular state. The effective concentration of the chain ends in the reaction is then 87 000-fold greater than in the random coil state. These results show that DNA globularity must be taken into account to understand biological processes involving intramolecular DNA-DNA interactions.
Subject(s)
Bacteriophage lambda/chemistry , DNA, Viral/chemistry , Nucleic Acid Conformation , Bacteriophage lambda/physiology , DNA, Circular/chemistry , DNA, Circular/drug effects , DNA, Viral/drug effects , DNA, Viral/physiology , Dose-Response Relationship, Drug , Nucleic Acid Conformation/drug effects , Spermidine/pharmacology , UltracentrifugationABSTRACT
In the presence of spermidine, the DNA molecule of the bacteriophage lambda undergoes a coil-globule transition. We report here that the cyclization of this molecule in its globular state is greatly accelerated (by more than 10(4)-fold) in comparison with the cyclization reaction taking place in the coil conformation.
