Rca reporter probes and their use in detecting nucleic acid molecules

A probe and molecule technology, applied in the field of detection of analytes such as target nucleic acid molecules and nucleic acid molecules, can solve the problems of increasing time and resources, difficulty in automation, etc.

Inactive Publication Date: 2015-09-30
OLINK AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Addition of components to an assay can also lead to an increase in the number of operational steps and, correspondingly, the amount of time and resources required to complete the assay, i.e. additional components can make automation of the assay more difficult

Method used

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  • Rca reporter probes and their use in detecting nucleic acid molecules
  • Rca reporter probes and their use in detecting nucleic acid molecules
  • Rca reporter probes and their use in detecting nucleic acid molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0314] Example 1 - Two-Part Hairpin RCA Probes

[0315] As noted above, the probes of the invention can be used to detect any analyte wherein an intermediate binding partner that can bind to the analyte is coupled to (or comprises) a nucleic acid molecule. A nucleic acid molecule coupled to an intermediate molecule, which serves as a surrogate or marker for the analyte, can be detected using the probe. The protocol described hereinafter uses a proximity probe as an intermediate binding molecule to a target analyte, wherein the target complementary domain of the RCA probe binds to the nucleic acid domain of the proximity probe (see e.g. figure 2). RCA probes were compared to padlock probes using gap oligonucleotides that are also capable of binding to the nucleic acid domains of the proximity probes to generate RCA templates. The RCA reaction of the padlock probe RCA template is initiated by one of the proximity probe nucleic acid domains.

[0316] 50 μl of biotinylated m...

Embodiment 2- 3

[0323] Example 2 - Three-Part Hairpin RCA Probe

[0324] The utility of "triple-part" RCA probes was demonstrated using nucleic acid analytes, ie, the target analyte is a nucleic acid molecule. Two variants of "triple-part" probes were tested, namely single-stranded probes containing 3 hairpin structures and partially double-stranded probes containing hairpin structures and two cleaved strands.

[0325] 50 μl of 10 pM synthetic biotinylated DNA template was incubated on streptavidin-coated slides (SurModics) in 1×PBS at 37° C. for 60 minutes, with 1×PBS without DNA incorporated as a negative control. After incubation, block with blocking buffer (0.1% BSA (Sigma), 100nM goat IgG (Sigma), 1mM biotin (Sigma), 10ng / μl salmon sperm DNA (Sigma), 5mM EDTA, 1x PBS and 0.05% Tween 20) Slides were blocked at 37°C for 60 minutes.

[0326] Streptavidin-coated slides were compartmentalized with secure-Seal 8 (Grace Bio-labs) prior to the experiment. A wash step (with two replicates of...

Embodiment 3

[0331] Continuous two-part probe and cut strand two-part probe in the detection of embodiment 3-nucleic acid target

[0332] The utility of "two-part" RCA probes is demonstrated using nucleic acid analytes, ie the target analyte is a nucleic acid molecule. Target nucleic acids were immobilized on glass slides using two different techniques. Two variants of "two-part" probes were tested, namely a continuous probe comprising two hairpin structures and a partially double-stranded probe comprising both a hairpin structure and a cleaved strand.

[0333] Immobilization of synthetic DNA templates

[0334] 10 [mu]l of synthetic biotinylated DNA template in 1x PBS was exposed to streptavidin-coated slides (SurModics) for 10 min at 55[deg.]C. Alternatively, hybridize the DNA template to the slide in 50 μl 1x PBS for 60 minutes at 37°C, using 1x PBS as a negative control. After incubation, at 37 ° C with blocking buffer (0.1% BSA (Sigma), 100nM goat IgG (Sigma), 1mM biotin (Sigma), ...

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Abstract

The present invention provides a probe for use in detecting a target analyte in a sample, wherein the probe provides or is capable of providing nucleic acid components sufficient to initiate a rolling circle amplification (RCA) reaction, said probe being a nucleic acid construct comprising: (i) one or more target binding domains capable of binding to said target analyte or an intermediate molecule bound, directly or indirectly, to the target analyte; (ii) one or more domains together comprising or capable of providing a circular or circularisable RCA template; (iii) a domain comprising or capable of providing a primer for said RCA reaction that hybridizes to a region of said circular or circularisable RCA template; and,when the probe comprises or is capable of providing a circularisable RCA template, (iv) one or more domains comprising or capable of providing a ligation template that templates the ligation of the circularisable RCA template, wherein at least part of the probe must be cleaved and / or unfolded to release said primer to enable said rolling circle amplification reaction. Also provided are methods for detecting analytes in a sample using the probe of the invention. In certain preferred embodiments of the probe cleavage of the probe into multiple parts each held in proximity by a target binding domain in each part of the probe generates a circularisable RCA template, which is circularised in a ligation reaction templated by a ligaton template domain of the probe.

Description

technical field [0001] The present invention relates to the detection of the analyte in the sample, relate in particular to the detection of the nucleic acid molecule (for example nucleic acid analyte) in the sample, and described analyte can be the analyte to be detected or can indicate the presence of the analyte in the sample ( That is, a marker or surrogate for the analyte). The present invention relates to the provision of novel nucleic acid detection probes (nucleic acid constructs) for use in rolling circle amplification (RCA) assays, also known as rolling circle replication (RCR) assays. The invention also provides methods of using the probes for the detection of analytes, such as target nucleic acid molecules (ie, nucleic acid analytes). [0002] The detection probes of the invention are designed to provide a nucleic acid component (i.e., a substrate) sufficient to initiate (i.e., directly enable) the RCA (RCR) reaction (i.e., without adding an additional nucleic aci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q1/682C12Q1/6848C12Q1/6804C12Q1/6853C12Q2521/501C12Q2525/131C12Q2525/301C12Q2525/307C12Q2531/125C12Q2521/531
Inventor 乌尔夫·兰德格伦陈磊吴迪农园
Owner OLINK AB
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