Spatial Genetic Variant Detection via Reversible Terminator Ligation
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Solution Overview
Problem
Existing methods for spatially detecting genetic variants in tissues fail to provide accurate information about the position of single cells within a biological sample, particularly when the variants are near the 5′ end of the target nucleic acid, and current templated-ligation methods suffer from non-specific binding and low specificity.
Innovation Solution
The use of templated-ligation with reversible terminator nucleotides, where a first probe hybridizes to a target nucleic acid and is extended with a reversible terminator nucleotide, followed by hybridization and ligation with a second probe, enhancing specificity through the use of polymerases like Bst 3 or Klenow polymerase and ligases like PBCV-1 ligase, to detect genetic variants such as single-nucleotide polymorphisms, insertions, and deletions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional templated-ligation methods are used for spatial detection of genetic variants, then the method can detect genetic variants in tissue samples, but the specificity is low due to non-specific binding of probes near genetic variants
Solution Approach 1:
The probe is divided into multiple segments: a first probe that hybridizes upstream of the genetic variant and a second probe that hybridizes downstream. This segmentation allows each probe to bind to stable regions away from the variant site, reducing non-specific binding while maintaining detection capability through ligation of the two probes.
Solution Approach 2:
A polymerase enzyme is introduced as an intermediary to extend the first probe by one nucleotide before ligation with the second probe. This intermediary step provides an additional specificity checkpoint, as the polymerase only extends when the first probe is correctly positioned, thereby reducing non-specific binding events.
2Quantity of substance
If poly(T) capture probes are used for spatial analysis, then a high number of analytes can be detected, but off-target analytes are also detected reducing accuracy
Solution Approach 1:
The detection system is segmented into multiple components: a poly(T) capture probe for high-capacity binding, flanked by specific genomic sequences. The requirement for ligation of additional probes with specific sequences provides a second layer of specificity, allowing high quantity detection while maintaining precision through the multi-step verification process.
3Measurement precision
If probe pairs are designed for specific target nucleic acids to increase specificity, then detection accuracy improves, but the method becomes less effective for variants near the 5′ end of the target
Solution Approach 1:
Instead of designing probes that start at or near the 5′ end of the target sequence, the approach inverts the strategy by designing the first probe to bind upstream and the second probe to bind downstream of the variant. This inversion allows detection of variants near the 5′ end by approaching from both directions and meeting at the variant site through ligation.
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
This approach increases the specificity and efficiency of detecting genetic variants by forming stable ligation products that can be captured on a spatial array, allowing for high-resolution spatial analysis of genetic variants within biological samples.
Implementation Method 1
a first probe of the plurality of first probes includes a sequence substantially complementary to a sequence of a target nucleic acid in the biological sample; hybridizing the first probe to the target nucleic acid
Implementation Method 2
extending the first probe using a reversible terminator nucleotide, thereby generating an extended first probe
Implementation Method 3
ligating the extended first probe to the second probe, thereby generating a ligation product
Data Source
AI summary
Provided herein are methods, compositions, and kits to spatially detect genetic variants in a target nucleic acid via templated-ligation and reversible terminator nucleotides.