Hyperthermophilic polymerase enabled proximity extension assay
Inventors
Fredriksson, Simon • Lundberg, Martin • Eriksson, Anna • Rennel-Dickens, Emma
Assignees
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Publication Number
US-9902993-B2
Publication Date
2018-02-27
Expiration Date
Abstract
The present invention relates to a proximity probe based detection assay (“proximity assay”) for an analyte in a sample, specifically a proximity probe extension assay (PEA), an in particular to an improvement in the method to reduce non-specific “background” signals, wherein the improvement comprises the use in such assays of a hyperthermophilic polymerase, said method comprising: (a) contacting said sample with at least one set of at least first and second proximity probes, which probes each comprise an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte; (b) allowing the nucleic acid domains of the proximity probes to interact with each other upon binding of said proximity probes to said analyte, wherein said interaction comprises the formation of a duplex; (c) extending the 3′ end of at least one nucleic acid domain of said duplex to generate an extension product, wherein the extension reaction comprises increasing the temperature of assay above room temperature and uses a polymerase enzyme which is characterised as having less than 20% of its maximal enzyme activity at 40° C. and having less than 10% of its maximal enzyme activity at 25° C., wherein the optimum temperature for maximal activity of the polymerase is more than 40° C. and wherein the polymerase is selected from Pyrococcus furiosus (Pfu) DNA polymerase and Pyrococcus woesei (Pwo) DNA polymerase or a derivative or mutant thereof, preferably wherein said derivative is a sequence-modified derivative; and (d) amplifying and detecting the extension product.
Core Innovation
The invention relates to a proximity probe extension assay (PEA) method for detecting an analyte in a sample. The method uses at least one set of proximity probes in which each probe comprises an analyte-binding domain and a nucleic acid domain that simultaneously bind to the analyte, and the nucleic acid domains interact to form a duplex.
A polymerase enzyme is contacted with the sample when the sample has less than or equal to 20% of maximal enzyme activity at 40°C and less than or equal to 10% of maximal enzyme activity at 25°C, with an optimum temperature for maximal activity more than 40°C. During the contacting step, the temperature of the sample is less than 40°C so that the polymerase enzyme has no or minimal activity, followed by increasing the temperature above the temperature of that step to increase polymerase activity and extend the 3′ end of at least one nucleic acid domain of the duplex to generate an extension product.
The extension product is then amplified and detected, with documented improvements directed to reducing non-specific background and improving signal-to-noise. The description further identifies optional background-reduction approaches including adding 3′ exonuclease activity components, using unfolding (hairpin) proximity probes, and using duplex/splint mediated probe interactions, as well as detection features such as qPCR with intercalating dyes including SYBR Green and EvaGreen.
Claims Coverage
The independent claim is directed to detecting an analyte using proximity probes that form a duplex, followed by controlled 3′ end extension using a polymerase selected for minimal activity at sub-40°C temperatures and subsequent temperature increase, then amplification and detection. The claim includes four inventive features.
Duplex-forming proximity probes for analyte detection
At least one set of proximity probes that each comprise an analyte-binding domain and a nucleic acid domain that can simultaneously bind to the analyte, wherein the nucleic acid domains interact upon binding to form a duplex.
Hyperthermophilic polymerase with defined low activity thresholds at 40°C and 25°C
Contacting the sample with a polymerase enzyme selected from Pyrococcus furiosus (Pfu) DNA polymerase and Pyrococcus woesei (Pwo) DNA polymerase, or a derivative or mutant thereof, having less than or equal to 20% of its maximal enzyme activity at 40°C and less than or equal to 10% of its maximal enzyme activity at 25°C, with an optimum temperature for maximal activity more than 40°C and having no or minimal activity during contacting when the sample temperature is less than 40°C.
Controlled temperature increase to activate extension and extend the 3′ end
After the low-temperature contacting, increasing the temperature of the resulting assay above the temperature of the contacting step so that increasing temperature increases polymerase activity, and extending the 3′ end of at least one nucleic acid domain of the duplex to generate an extension product using the polymerase enzyme.
Amplifying and detecting the extension product
Amplifying and detecting the extension product.
Across the independent claim, the core coverage centers on duplex formation by proximity probes, controlled activation of 3′ extension using a polymerase with defined low activity at 40°C and 25°C and optimum above 40°C, and subsequent amplification and detection of the extension product.
Stated Advantages
Reduces non-specific background.
Improves specificity/sensitivity via minimal polymerase activity during a low-temperature phase before extension.
Enables simpler closed-tube workflows.
Supports quantitative detection by fluorescence monitoring, including melting-peak analysis with intercalating dyes such as SYBR Green or EvaGreen.
Enables multiplex analysis using multiple sets of proximity probes that generate distinct extension products.
Reduces background in proximity extension assays through use of single-strand DNA binding proteins, added RNA, partially double-stranded nucleic acid domains, and blocking or competitor oligonucleotides.
Documented Applications
Proximity probe extension assay (PEA) for detecting an analyte in a sample.
Assays using qPCR detection with intercalating dyes including SYBR Green and EvaGreen.
Detecting analytes in a sample using proximity extension assays with fluorescence monitoring and melting-peak analysis for real-time or quantitative PCR readouts, including examples using interleukin-8 (IL-8), VEGF, and GDNF.
Multiplex analysis in proximity extension assays where multiple probe sets generate distinct extension products.
Solid-phase readout formats using DNA array hybridization tags and hybridization-based detection of proximity extension products.
Use of polymerase extension and amplification and detection workflows in homogeneous and solid-phase assay formats.
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