Padlock Probe In Situ Assays for Specific SNP Detection
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Solution Overview
Problem
Existing methods for analyzing short sequences such as single nucleotide polymorphisms (SNPs) or point mutations in nucleic acids, particularly in situ, suffer from low sensitivity, specificity, and complexity, with challenges in distinguishing single bases and requiring labor-intensive processes.
Innovation Solution
A method involving the use of control and detection padlock probes, anchors, and rolling circle amplification to form circularized probes, allowing for the generation and detection of amplification products that indicate the presence or absence of specific nucleic acid regions, including SNPs, through hybridization and ligation processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional in situ hybridization methods are used for analyzing short nucleic acid sequences, then spatial information is preserved, but sensitivity and specificity are low
Solution Approach 1:
The patent introduces padlock probes as intermediary molecules that bridge the target nucleic acid and the detection system. These probes hybridize to the target sequence and can be circularized and amplified, serving as a mediator that enhances both sensitivity and specificity of detection while preserving spatial information in situ
Solution Approach 2:
The method performs preliminary hybridization of padlock probes to target nucleic acids before amplification. This preliminary binding step ensures specific recognition of the target sequence, and subsequent circularization and rolling circle amplification amplify only the specifically bound probes, thereby improving both sensitivity and specificity
2Productivity
If traditional methods are used for in situ SNP genotyping, then spatial information is maintained, but the process is labor-intensive and complex
Solution Approach 1:
The patent combines multiple functions into a single integrated assay system. The padlock probes simultaneously perform target recognition, signal amplification through rolling circle amplification, and spatial preservation. This merging of functions reduces the number of separate steps required, thereby increasing throughput while managing complexity
Solution Approach 2:
The method uses rolling circle amplification to generate multiple copies of the padlock probe signal. This copying mechanism amplifies the detection signal from each bound probe, enabling high-throughput detection without requiring complex instrumentation or multiple separate assay steps
3Measurement precision
If signal amplification is performed to improve detection sensitivity, then short sequences can be detected, but false positives increase and specificity decreases
Solution Approach 1:
The patent implements a feedback mechanism through controlled circularization of padlock probes. Only probes that are correctly hybridized to the target can be circularized and amplified, creating a feedback loop that ensures amplification occurs only for specifically bound probes. This reduces false positives while maintaining sensitivity through signal amplification
Solution Approach 2:
The padlock probe serves as an intermediary that must undergo successful hybridization and circularization before amplification can occur. This multi-step intermediary process acts as a filter, ensuring that only specifically bound probes are amplified, thereby reducing false positives while maintaining high detection sensitivity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables high-throughput, sensitive, and specific detection and analysis of short nucleotide sequences in situ, preserving spatial information in biological samples without physical isolation, with improved signal-to-noise ratios and reduced labor intensity.
Implementation Method 1
contacting the target nucleic acid with a control padlock probe and a detection complex comprising a detection padlock probe... the detection complex comprises a sequence complementary to the region of interest and the control padlock probe comprises a sequence complementary to the target nucleic acid
Implementation Method 2
forming a circularized control padlock probe by ligating the control padlock probe using the anchor as a template
Implementation Method 3
generating an amplification product using the circularized detection padlock probe as a template and an amplification product using the circularized control padlock probe as a template
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 2C~2D
AI summary
The present disclosure relates in some aspects to methods for analyzing a target nucleic acid in a biological sample. In some aspects, the methods involve the use of a set of polynucleotides, including one or more polynucleotides (e.g., a detection padlock probe) for detecting a region of interest and one or more polynucleotides (e.g., a control padlock probe) as an internal control, for analyzing target nucleic acids. In some aspects, the presence, amount, and/or identity of a region of interest in a target nucleic acid is analyzed in situ. Also provided are polynucleotides, sets of polynucleotides, compositions, and kits for use in accordance with the methods.