Abstract
Detection of low frequency single nucleotide polymorphisms (SNPs) has important implications in early screening for tumorgenesis, genetic disorders and pathogen drug resistance. Nucleic acid arrays are a powerful tool for genome-scale SNP analysis, but detection of low-frequency SNPs in a mixed population on an array is problematic. We demonstrate a model assay for HIV-1 drug resistance mutations, wherein ligase discrimination products are collected on a suspension array. In developing this system, we discovered that signal from multiple polymorphisms was obscured by two discrete hybridization artifacts. Specifically: 1) tethering of unligated probes on the template DNA elicited false signal and 2) unpredictable probe secondary structures impaired probe capture and suppressed legitimate signal from the array. Two sets of oligonucleotides were used to disrupt these structures; one to displace unligated reporter labels from the bead-bound species and another to occupy sequences which interfered with array hybridization. This artifact silencing system resulted in a mean 21-fold increased sensitivity for 29 minority variants of 17 codons in our model assay for mutations most commonly associated with HIV-1 drug resistance. Furthermore, since the artifacts we characterized are not unique to our system, their specific inhibition might improve the quality of data from solid-state microarrays as well as from the growing number of multiple analyte suspension arrays relying on sequence-specific nucleic acid target capture.
Highlights
Assays for single nucleotide polymorphisms (SNPs) are useful for haplotyping [1], identifying pathogen species [2], and screening for heritable polymorphisms [3]
A Multiplex Assay for Drug Resistant Mutations in HIV-1 Pol Five classes of antiretroviral drugs, i.e. protease inhibitors (PIs), nucleoside analogue reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), integrase inhibitors (INIs) and entry inhibitors, are commonly prescribed as part of highly active antiretroviral treatment regimen, but discrete mutations conferring resistance are observed for only the PI, NRTI, NNRTI, and INI drug classes
Subsequent flow fluorimetry assesses reporter enrichment for each bead region and, since the reporter fluorophore should only associate with beads via RCO linkage to the BCO, reporter associated with a specific bead region reflects how much of the region-associated SNP was present in the template DNA
Summary
Assays for single nucleotide polymorphisms (SNPs) are useful for haplotyping [1], identifying pathogen species [2], and screening for heritable polymorphisms [3]. Target molecules are captured on optically identifiable beads and quantified by a flow fluorimeter, an instrument equivalent to a three-color flow cytometer with dedicated gating for beads in suspension [6]. This system, equivalent to a solid-state array in many capacities, can be used to simultaneously detect multiple proteins [7] or nucleic acids [2] via identifiable fluorescent signatures on beads with a corresponding affinity label. Bead-captured BCO-RCO ligation products are labeled with a streptavidin-Rphycoerythrin conjugate (the reporter fluorophore), enriching beads with the reporter in proportion to ligase joining. Each bead is identified (indicating what SNP is being detected) and evaluated for reporter fluorophore enrichment (indicating how much of that SNP was present) in a liquid flow as they pass through the optical chamber in the flow fluorimeter
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