PNA Probe Capture of Long DNA Fragments for Methylation Phasing
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Solution Overview
Problem
Existing methods struggle to efficiently isolate and sequence long double-stranded DNA fragments for constructing phased haplotypes while preserving methylation status, as they often result in DNA methylation information loss and imbalanced allele ratios.
Innovation Solution
The use of peptide nucleic acid (PNA) hybridization probes with capture tags and modified backbones to target and enrich long DNA fragments, allowing for sequence-specific capture and sequencing that preserves methylation patterns and maintains allele balance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If solution DNA capture using DNA or RNA probes is used, then genomic regions can be captured, but long single-stranded DNA molecules undergo intermolecular re-association forming DNA polymer networks that prevent specific target isolation
Solution Approach 1:
The patent introduces a double-stranded DNA intermediate molecule that acts as a mediator between the single-stranded genomic DNA and the capture probes. The ssDNA first hybridizes to the complementary strand of the dsDNA intermediate, forming a stable duplex. This intermediate structure prevents direct intermolecular re-association of ssDNA molecules while providing a controlled platform for specific probe binding, thereby resolving the contradiction between maintaining ssDNA integrity and preventing harmful polymer network formation.
2Length of moving object
If fosmid cloning is used to enrich long genomic DNA domains, then long DNA can be isolated, but DNA methylation information is lost and the process is time-consuming
Solution Approach 1:
The patent replaces the mechanical cloning process (fosmid vector insertion, bacterial transformation, and plasmid extraction) with a direct in-solution hybridization and capture system. Genomic DNA is sheared to appropriate lengths, hybridized to dsDNA intermediates, and then captured using biotinylated probes followed by streptavidin bead selection. This substitution eliminates the cloning step entirely, preserving native DNA modifications including methylation while significantly reducing processing time.
3Productivity
If ligation-mediated PCR is used in PacBio sequencing library preparation, then long reads can be obtained, but imbalances in the ratio of maternal and paternal alleles occur
Solution Approach 1:
The patent extracts and eliminates the LMP step from the library preparation workflow. Instead of using LMP to generate long reads, the method directly captures long genomic DNA fragments (10-100 kb) using dsDNA intermediate-mediated hybridization and affinity capture. The captured long fragments are then directly used for sequencing library construction, bypassing PCR amplification entirely. This extraction of the problematic LMP step preserves the natural allele representation while maintaining long read capability.
4Quantity of substance
If conventional DNA capture methods are used, then sequence-specific enrichment can be achieved, but long DNA fragments greater than 2 kilobases cannot be effectively isolated while preserving native structure and methylation status
Solution Approach 1:
The patent performs preliminary hybridization of genomic DNA to complementary dsDNA intermediates before any capture or processing steps. This preliminary action creates stable duplex structures that protect the long DNA fragments from degradation and preserve their native conformation. The dsDNA intermediate acts as a protective scaffold that maintains fragment integrity throughout subsequent probe binding and affinity capture steps, ensuring that long fragments retain their native structure and epigenetic modifications.
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
Enriches long DNA fragments (2,000 to 40,000 bases) for sequencing, enabling accurate phasing of DNA methylation patterns and identifying short insertions/deletions, with improved specificity and efficiency compared to conventional methods.
Implementation Method 1
PNA hybridization probes with capture tags and modified backbones to target and enrich long DNA fragments
Data Source
AI summary
It has been established that one or more large double stranded DNA fragments (each 2,000 to 40,000 base pairs in size) can be captured and isolated from genomic DNA fragments using sequence specific PNA hybridization probes. Compositions and methods for enrichment of a multiplicity of long DNA sequences selected from the genome of any eukaryote are provided. Capture is performed using multiple PNA molecules with gamma-modified chiral backbones, comprising a mixture of neutral and positive chemical groups. Two or more PNA probes with covalently bound haptens, preferably biotin, target each DNA domain of interest for capture, isolation, and subsequent sequencing analysis of the multiplicity of enriched targets, including DNA methylation sequencing. The methods include enhancement of probe-DNA binding specificity through single strand binding proteins (SSB).