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• W2798104660 abstract "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– 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." @default.
• W2798104660 created "2018-04-24" @default.
• W2798104660 creator A5045893467 @default.
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• W2798104660 date "2018-04-13" @default.
• W2798104660 modified "2023-10-17" @default.
• W2798104660 title "Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification" @default.
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• W2798104660 doi "https://doi.org/10.1021/acs.analchem.8b00058" @default.
• W2798104660 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/29653055" @default.
• W2798104660 hasPublicationYear "2018" @default.
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