Targeted Expansion Sequencing Protocols v2

This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry. For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).

Targeted Expansion Sequencing Protocols v3

This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry. For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).

Designing a fluorescence padlock probe-based biosensor and colorimetric assay for the detection of G12D KRAS mutation

Aim: Cell-free DNA in the plasma is known to be a potential biomarker for noninvasive diagnosis of oncogenic mutations. The authors aimed to design an optimized padlock probe-based hyperbranched rolling circle amplification biosensor to detect the KRAS G12D mutation using fluorescence and colorimetric methods. Methods: Single-factor experiments, Plackett–Burman design and response surface methodology were applied to optimize the padlock probe-based hyperbranched rolling circle amplification reaction. Results: The maximum fluorescence intensity was achieved at a padlock probe concentration of 1.5 pM and target concentration of 9 pM at 38°C ligation temperature. The proposed biosensor has a low detection limit of 60 fM of target DNA and a linear response in the concentration range of 60 fM to 0.2 pM. Conclusion: The results indicated the power of these assays to detect KRAS point mutations in liquid state reactions.

MiRNA Detection Using a Rolling Circle Amplification and RNA-Cutting Allosteric Deoxyribozyme Dual Signal Amplification Strategy

A microRNA (miRNA) detection platform composed of a rolling circle amplification (RCA) system and an allosteric deoxyribozyme system is proposed, which can detect miRNA-21 rapidly and efficiently. Padlock probe hybridization with the target miRNA is achieved through complementary base pairing and the padlock probe forms a closed circular template under the action of ligase; this circular template results in RCA. In the presence of DNA polymerase, RCA proceeds and a long chain with numerous repeating units is formed. In the presence of single-stranded DNA (H1 and H2), multi-component nucleic acid enzymes (MNAzymes) are formed that have the ability to cleave substrates. Finally, substrates containing fluorescent and quenching groups and magnesium ions are added to the system to activate the MNAzyme and the substrate cleavage reaction, thus achieving fluorescence intensity amplification. The RCA–MNAzyme system has dual signal amplification and presents a sensing platform that demonstrates broad prospects in the analysis and detection of nucleic acids.

Padlock Probe-based Rolling Circle Amplification Lateral Flow Assay for Point-of-Need Nucleic Acid Detection

Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs)...