4C Genomic Interaction Analysis via High-Throughput Sequencing
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Solution Overview
Problem
Current 3C and 4C technologies are limited in their ability to systematically analyze the whole genome for DNA-DNA interactions, particularly for long-range interactions and genomic rearrangements such as translocations, inversions, deletions, and amplifications, due to limitations in PCR amplification and the need for specific sequence information, which restricts their high-throughput capability and unbiased screening.
Innovation Solution
The method involves high-throughput sequencing to analyze DNA interactions, allowing for the simultaneous analysis of multiple target sequences and the identification of genomic rearrangements by sequencing the ligation junctions between target and captured sequences, providing absolute numbers and overcoming the limitations of microarray saturation and the need for specific primer design.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR amplification is used to analyze DNA interactions, then specific sequence information can be obtained, but the ability to systematically analyze the whole genome is limited
Solution Approach 1:
The patent replaces the PCR amplification mechanism with high-throughput sequencing technology. Instead of using polymerase chain reaction to amplify and detect specific DNA sequences, the invention employs next-generation sequencing platforms to directly sequence ligation junctions between cross-linked DNA fragments. This substitution enables simultaneous analysis of thousands of DNA interaction sites across the entire genome, transforming the method from targeted analysis to comprehensive genome-wide screening while maintaining the ability to identify specific interaction sequences.
2Reliability
If specific primer design is required for PCR analysis, then targeted DNA fragments can be analyzed, but unbiased screening of the entire genome is restricted
Solution Approach 1:
The patent extracts the requirement for prior sequence knowledge and primer design from the analysis workflow. By using high-throughput sequencing to directly read the sequences at ligation junctions, the method eliminates the need to pre-design primers for each DNA fragment of interest. The sequencing technology automatically determines the identity of interacting DNA sequences based on their sequence reads, enabling unbiased discovery of novel interactions without requiring researchers to specify target sequences in advance.
3Measurement precision
If microarray hybridization is used to detect DNA interactions, then interaction frequencies can be measured, but the dynamic range is limited by saturation
Solution Approach 1:
The patent replaces microarray hybridization with high-throughput sequencing-based quantification. Instead of measuring interaction frequencies through hybridization signal intensities that are prone to saturation, the invention counts the number of sequencing reads originating from each ligation junction. This digital counting approach provides a linear relationship between read depth and interaction frequency across a much wider dynamic range, allowing accurate measurement of both highly frequent and rare DNA interactions without signal saturation.
4Reliability
If conventional 3C/4C technology is used, then DNA interaction analysis is possible, but high-throughput capability and resolution are insufficient
Solution Approach 1:
The patent fundamentally changes the detection parameter from low-throughput PCR amplification or microarray hybridization to high-throughput sequencing. This parameter change increases the analytical capacity by several orders of magnitude, enabling the simultaneous analysis of thousands of DNA interaction sites across the entire genome. The sequencing approach provides base-pair resolution of interaction sites and can detect both common and rare interaction events, dramatically improving both the scale and resolution of genomic interaction analysis compared to conventional 3C and 4C methodologies.
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 enables high-throughput, unbiased genome-wide screening for DNA-DNA interactions and genomic rearrangements, increasing the scale and resolution of analysis, and allowing for the detection of balanced and unbalanced genetic aberrations, even when the bait sequence is located far from the rearrangement site, facilitating early disease diagnosis and prognosis.
Implementation Method 1
providing a sample of cross-linked DNA
Implementation Method 2
digesting the cross-linked DNA with a primary restriction enzyme
Implementation Method 3
ligating the cross-linked nucleotide sequences
Implementation Method 4
amplifying the one or more nucleotide sequences of interest using at least two oligonucleotide primers
Implementation Method 5
The amplified sequences can be hybridised to an array in order to assist in determining the frequency of interaction between the DNA sequences
Data Source
Figure 1
Figure 2a~2b
Figure 3
AI summary
The present invention relates in one aspect to a method for analysing the frequency of interaction of a target nucleotide sequence with one or more nucleotide sequences of interest (eg. one or more genomic loci) comprising the steps of: (a) providing a sample of cross-linked DNA; (b) digesting the cross-linked DNA with a primary restriction enzyme; (c) ligating the cross-linked nucleotide sequences; (d) reversing the cross linking; (e) optionally digesting the nucleotide sequences with a secondary restriction enzyme; (f) optionally ligating one or more DNA sequences of known nucleotide composition to the available secondary restriction enzyme digestion site(s) that flank the one or more nucleotide sequences of interest; (g) amplifying the one or more nucleotide sequences of interest using at least two oligonucleotide primers, wherein each primer hybridises to the DNA sequences that flank the nucleotide sequences of interest; (h) hybridising the amplified sequence(s) to an array; and (i) determining the frequency of interaction between the DNA sequences.