Cleavable Nucleic Acid Moieties for Sequencing Accuracy
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Solution Overview
Problem
Current nucleic acid sequencing methods face challenges with single-stranded DNA molecules due to cross-hybridization, bead clumping, and loss of genome coverage, particularly because of repetitive sequences and secondary structure formation, which impede accurate and efficient sequencing.
Innovation Solution
The use of double-stranded nucleic acid molecules linked to solid supports with cleavable bases, allowing for methods to isolate, enrich, and sequence these molecules by excising cleavable bases, enabling analysis and sequencing through specific enzymatic treatments and polymerase interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If single-stranded DNA molecules are used for sequencing, then sequencing can be performed on beads with clonal populations, but cross-hybridization occurs leading to bead clumping and loss of genome coverage
Solution Approach 1:
The invention uses double-stranded DNA molecules instead of single-stranded molecules, effectively segmenting the problematic single strand into two separate strands that can be independently managed. This prevents cross-hybridization between complementary sequences and eliminates bead clumping while maintaining sequencing throughput
Solution Approach 2:
The invention introduces a secondary strand as an intermediary that protects the primary sequencing strand from unwanted interactions. The double-stranded structure acts as a mediator that prevents direct cross-hybridization between different bead-bound molecules, thereby maintaining genome coverage accuracy
2Ease of manufacture
If single-stranded molecules are used, then sequencing preparation can be simplified, but secondary structure formation impedes sequencing progression and generates context bias
Solution Approach 1:
Instead of working with single-stranded DNA that naturally forms secondary structures, the invention inverts the approach by using double-stranded DNA. This inversion prevents the formation of intra-strand secondary structures that cause sequencing bias, while the strands can be denatured into single strands for sequencing when needed
3Measurement precision
If cleavable bases are excised from double-stranded molecules, then sequencing accuracy is improved by reducing cross-hybridization, but additional processing steps are required
Solution Approach 1:
The invention extracts or removes cleavable bases from the double-stranded DNA molecules through enzymatic treatment. This extraction eliminates sources of sequencing error and cross-hybridization interference, improving sequencing accuracy while the removed bases can be discarded or recovered
Solution Approach 2:
The invention replaces mechanical or chemical cleavage methods with enzymatic treatment for removing cleavable bases. This substitution provides more specific and controlled base removal, improving sequencing accuracy while maintaining process efficiency
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 enhances sequencing accuracy and efficiency by reducing cross-hybridization and bead clumping, improving genome coverage and sequencing precision by working with double-stranded molecules and incorporating labeled nucleotides for signal detection.
Implementation Method 1
contacting the nucleic acid molecule with a cleaving agent configured to cleave or excise the one or more of the at least two cleavable or excisable moieties
Implementation Method 2
the labeled nucleotide is configured to emit a signal
Implementation Method 3
bringing the cleaved nucleic acid molecule into contact with a polymerase enzyme and a labeled nucleotide
Data Source
AI summary
The present disclosure provides compositions comprising nucleic acid molecules coupled to supports and comprising one or more cleavable or excisable moieties. Methods of enriching and sequencing nucleic acid molecules are also provided.