title: Conversion strategy using an expanded genetic alphabet to assay nucleic acids.
abstract: Methods to detect DNA and RNA (collectively xNA) are easily plagued by noise, false positives, and false negatives, especially with increasing levels of multiplexing in complex assay mixtures. Here, we describe assay architectures that mitigate these problems by converting standard xNA analyte sequences into sequences that incorporate nonstandard nucleotides (Z and P). Z and P are extra DNA building blocks that form tight nonstandard base pairs without cross-binding to natural oligonucleotides containing G, A, C, and T (GACT). The resulting improvements are assessed in an assay that inverts the standard Luminex xTAG architecture, placing a biotin on a primer (rather than on a triphosphate). This primer is extended on the target to create a standard GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of competition from any natural oligonucleotides, even in complex biological samples.
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), Gainesville, Florida 32601, United States. zyang@ffame.org
authors.forename: Zunyi
authors.initials: Z
authors.lastname: Yang
authors.forename: Michael
authors.initials: M
authors.lastname: Durante
authors.forename: Lyudmyla G
authors.initials: LG
authors.lastname: Glushakova
authors.forename: Nidhi
authors.initials: N
authors.lastname: Sharma
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Leal
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Bradley
authors.forename: Fei
authors.initials: F
authors.lastname: Chen
authors.forename: Steven A
authors.initials: SA
authors.lastname: Benner
mesh_terms: D006801:Humans; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017423:Sequence Analysis, RNA
chemical_list: D009696:Nucleic Acids
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