Nucleic Acid Construct Preparation via Transposase Insertion
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid sequencing technologies are slow and expensive due to reliance on amplification techniques and high quantities of specialist chemicals, necessitating the development of more efficient methods for polynucleotide characterization.
Innovation Solution
A method involving a polynucleotide-guided effector protein (PGEP) that directs a transposase to a specific region of a target polynucleotide, facilitating the insertion of a modified transposable element to enhance single molecule characterization, utilizing a system comprising a PGEP, a guide polynucleotide, a transposase, and a transposable element to prepare a nucleic acid construct for characterization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If amplification techniques are used to produce large volumes of polynucleotide, then signal detection capability is improved, but sequencing speed and cost efficiency deteriorate
Solution Approach 1:
The invention extracts and eliminates the amplification step from the sequencing workflow. By using nanopore detection that can directly sense single polynucleotide molecules, the method removes the need to produce large volumes of amplified DNA, thereby eliminating the time and cost associated with amplification while maintaining sufficient signal for detection
Solution Approach 2:
The invention introduces nanopores as an intermediary detection mechanism that bridges the gap between single molecules and detectable signals. The nanopores convert the presence and passage of individual polynucleotide molecules into measurable electrical current changes, enabling direct detection without amplification
2Quantity of substance
If amplification techniques are used to produce large volumes of polynucleotide, then signal detection capability is improved, but cost efficiency deteriorates
Solution Approach 1:
The invention extracts and eliminates the amplification step from the sequencing workflow. By using nanopore detection that can directly sense single polynucleotide molecules, the method removes the time and cost associated with amplification while maintaining sufficient signal for detection
Solution Approach 2:
The invention employs disposable nanopore sensors that can be used for single-molecule detection without requiring expensive amplification reagents or specialized fluorescent chemicals, reducing the overall cost of the sequencing process
3Productivity
If transposase activity is increased to improve insertion efficiency, then productivity is improved, but off-target activity increases
Solution Approach 1:
The invention introduces a programmable guide RNA as an intermediary that directs the transposase to specific target sequences. This guide RNA acts as a molecular address label, ensuring the transposase inserts the transposable element only at the intended location rather than randomly throughout the genome
Solution Approach 2:
The invention applies local quality by making the transposase activity site-specific through the guide RNA-target sequence pairing. The transposase is activated or directed only at the specific genomic location complementary to the guide RNA, creating localized precision rather than uniform activity across the entire genome
Data Source
AI summary
A method of preparing a nucleic acid construct for single molecule characterisation, comprising contacting a target polynucleotide with: a polynucleotide-guided effector protein, a guide polynucleotide; a transposase; and a transposable element comprising a modified polynucleotide, wherein the polynucleotide-guided effector protein directs said transposase to a region of interest within the target polynucleotide and the transposase inserts the transposable element into the polynucleotide, thereby producing a nucleic acid construct for single molecule characterisation.