4C Assay for Unbiased Genome-Wide DNA Interaction Screening
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Solution Overview
Problem
Current 3C technology is limited in its ability to systematically and unbiasedly analyze the frequency of interaction between DNA loci across the whole genome due to the requirement for specific sequence information and prior knowledge of hypersensitive sites, restricting its capability to screen for long-range DNA interactions.
Innovation Solution
The development of 4C technology, which modifies 3C by omitting the PCR step and using oligonucleotide primers that hybridize to flanking sequences, allowing for an unbiased genome-wide search for DNA fragments interacting with a target locus, and forming DNA circles to amplify interacting sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If 3C technology uses PCR amplification with specific primers to analyze DNA interactions, then measurement precision of specific DNA fragment interactions is improved, but productivity for genome-wide screening deteriorates due to requirement of prior sequence knowledge
Solution Approach 1:
The patent applies universality by designing a methodology that can analyze any DNA locus across the genome using the same general approach. Instead of requiring locus-specific primer design for each interaction, the method uses universal ligation-mediated amplification where primers bind to known flanking sequences, enabling the same protocol to screen the entire genome for interactions with any target locus.
Solution Approach 2:
The patent applies preliminary action by performing in vivo formaldehyde cross-linking and restriction enzyme digestion before amplification. The cross-linking step pre-establishes the spatial relationships between DNA loci, and the restriction digestion pre-prepares the DNA fragments for ligation, allowing the subsequent amplification step to simply detect pre-formed interaction products rather than requiring locus-specific optimization.
2Measurement precision
If 3C technology requires prior knowledge of hypersensitive sites and sequence information for PCR, then measurement precision of selected interactions is improved, but adaptability for unbiased genome-wide search deteriorates
Solution Approach 1:
The patent makes the methodology universal by using flanking sequence primers that can be designed once for any restriction site, rather than requiring locus-specific knowledge. The same general protocol works for any target locus in the genome, enabling unbiased screening without sacrificing the ability to precisely measure interactions at any specific location.
Solution Approach 2:
The patent inverts the traditional 3C approach by not starting with locus-specific primer design. Instead, it uses universal ligation-mediated amplification where the primers bind to known flanking sequences adjacent to restriction sites, and the interaction partners are captured through ligation to the target fragment. This reversal allows any genomic region to be analyzed without prior sequence knowledge of the interaction partners.
3Manufacturing precision
If 3C technology uses locus-specific PCR primers to analyze DNA interactions, then manufacturing precision of interaction analysis is improved, but device complexity for genome-wide application increases
Solution Approach 1:
The patent reduces system complexity by creating a universal amplification protocol that works for all loci. Instead of requiring separate optimized PCR conditions and primer designs for each genomic location, the same ligation-mediated amplification protocol with flanking sequence primers can be applied genome-wide, maintaining precision while eliminating the need for locus-specific optimization.
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
Enables high-throughput analysis of DNA-DNA interactions, detecting balanced or unbalanced genetic aberrations, and identifying predispositions to genomic rearrangements without requiring exact knowledge of change positions, facilitating the mapping of regulatory networks and disease-associated loci.
Implementation Method 1
using at least two oligonucleotide primers, wherein each primer hybridises to a DNA sequence that flanks the nucleotide sequences of interest
Implementation Method 2
ligation of DNA fragments that were cross-linked into one complex
Implementation Method 3
3C technology involves in vivo formaldehyde cross-linking of cells and nuclear digestion of chromatin
Data Source
Figure 1
Figure 2a~2c
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) digesting the nucleotide sequences with a secondary restriction enzyme; (f) 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.