Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification

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;

Split Dapoxyl Aptamer for Sequence-Selective Analysis of Nucleic Acid Sequence Based Amplification Amplicons

“…Therefore, nucleic acid amplification step is required prior to the analysis. We, therefore, turned our attention to NASBA, which is known to produce high concentrations of a single-stranded RNA amplicon, , and is considered one of the most promising isothermal amplification techniques. − A fragment of the ZIKV envelope gene was amplified by NASBA using RNA ZIKV (Brazil 1840 isolate) isolated from Vero cells to produce a 147-nt RNA amplicon ( Z-147 , Table S1). The production of Z-147 fragment by NASBA was confirmed by 2% agarose gel electrophoresis (Figure A).…”

Loop-Mediated Isothermal Amplification-Coupled Glass Nanopore Counting Toward Sensitive and Specific Nucleic Acid Testing

“…There have been a variety of molecule replication strategies developed. In addition to the traditional polymerase chain reaction (PCR), isothermal methods such as loop-mediated isothermal amplification (LAMP), , nucleic acid sequence-based amplification (NASBA), and recombinase polymerase amplification (RPA) have shown great promise for field use since they do not require thermocyclers and often are very fast.…”

Surface‐Bound Microgels for Separation, Sensing, and Biomedical Applications

“…For example, Ma et al developed a solid-phase oligonucleotide amplifier by integrating RNA primers on the surface of biotinylated-PEG microgels combined with reverse transcriptase and RNase H, which promoted the formation of double-stranded DNA on the microgel surface. [186] Teng et al used an electron beam to direct the surface patterning of DNA-presenting PEG microgels, demonstrating that co-localization of RNA primers alongside fluorescent molecular beacons on the microgel surface enabled DNA detection at concentrations as low as 10 -16 M. [187] Oligo-and poly-saccharides also offer valuable biological functionality, although their high hydrophilicity can dampen stimuli-responsiveness of the microgels, and their integration in microgels is more challenging than that of peptides and oligonucleotides. Tang et al prepared a set of glycomethacrylate monomers by conjugating lactose, melibiose, and fructose to methacryloyl chloride.…”