Capillary electrophoresis for capture and concentrating of target nucleic acids by affinity gels modified to contain single-stranded nucleic acid probes.
Selective capture and pre-concentration of target nucleic acids from relatively complicated samples may provide a method to facilitate introduction to a microfluidic-based detection system to improve detection limits. An acrylamide polymer gel modified with Acrydite that contained 20mer oligonucleotide probe was prepared and loaded into a capillary column. The results indicated that the amount of probe DNA that was captured into the acrylamide was about 40% of the starting monomer, and different quantities of probe could therefore be coupled into the gel. The gel was passivated by pre-treatment with non-complementary DNA oligonucleotide to block non-selective adsorption sites, and the gel was determined to be stable for multiple cycles of use. The probe could hybridize with target sequences that were introduced by electrokinetic injection from a sample solution. The target could be freed from the polymer gel by use of a combination of heating, chaotropic salt and voltage conditions. Target capture efficiency was up to 90% when using samples that did not saturate probe sites in the columns, and recovery of target from the gel could be as high as 95%.
Chan
Andrew
A
Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto at Mississauga, Mississauga, Ontario, Canada L5L 1C6.
Krull
Ulrich J
UJ