6 research outputs found
Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification
Solid-phase
oligonucleotide amplification is of interest because
of possible applications to next-generation sequencing, multiplexed
microarray-based detection, and cell-free synthetic biology. Its efficiency
is, however, less than that of traditional liquid-phase amplification
involving unconstrained primers and enzymes, and understanding how
to optimize the solid-phase amplification process remains challenging.
Here, we demonstrate the concept of solid-phase nucleic acid sequence-based
amplification (SP-NASBA) and use it to study the effect of tethering
density on amplification efficiency. SP-NASBA involves two enzymes,
avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase
H, to convert tethered forward and reverse primers into tethered double-stranded
DNA (ds-DNA) bridges from which RNA<sup>–</sup> amplicons can
be generated by a third enzyme,
T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam
patterning of thin-film blends of hydroxyl-terminated and biotin-terminated
poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly
related to the PEG-B concentration, and biotinylated primers and molecular
beacon detection probes are tethered to streptavidin-activated microgels.
While SP-NASBA is very efficient at low tethering densities, the efficiency
decreases dramatically with increasing tethering density due to three
effects: (a) a reduced hybridization efficiency of tethered molecular
beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency;
(c) inhibition of T7 RNA polymerase activity by AMV-RT