Twin Adapter Tagging for Restriction Library Self-Ligation
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Solution Overview
Problem
Traditional methods for tagging genomic DNA restriction libraries suffer from inaccuracies due to self-ligation of restriction fragments, which interferes with the accurate measurement of DNA methylation levels and comparison between samples.
Innovation Solution
The method involves using a twin adapter with a recognition site for a first restriction enzyme, digesting it to create adapters with the same restriction fragment ends as a second restriction enzyme, and then ligating these adapters to genomic DNA fragments, repeating the digestion and ligation process to eliminate self-ligation products and produce a library that is substantially free of self-ligation products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional tagging methods are used for restriction libraries, then the ligation process is simple, but self-ligation of restriction fragments occurs causing inaccuracies in methylation level measurement
Solution Approach 1:
The patent introduces an intermediary restriction site and adapter sequence between the original restriction fragment and the tagging sequence. The adapter contains a recognition site for a second restriction enzyme that is different from the first enzyme. This intermediary structure allows the fragment to be tagged while preventing self-ligation, because the adapter sequence disrupts the complementary pairing between identical fragment ends.
Solution Approach 2:
The tagging process is segmented into multiple distinct steps: (1) digestion with first restriction enzyme to create initial fragments, (2) ligation of adapter containing second restriction site, (3) digestion with second restriction enzyme, and (4) final ligation of tagging sequence. This segmentation allows each step to be optimized and controlled separately, improving overall accuracy while managing complexity.
2Productivity
If restriction fragments are digested and ligated to create a library, then the library can be amplified, but self-ligation products are generated that interfere with accurate analysis
Solution Approach 1:
The patent converts the potentially harmful self-ligation event into a beneficial filtering mechanism. By designing the adapter to contain a second restriction site, self-ligated fragments are created with two identical second restriction sites. These self-ligated products are then specifically removed by digesting with the second restriction enzyme, converting the harmful self-ligation into a useful way to identify and eliminate unwanted products.
Solution Approach 2:
The method implements a feedback mechanism where the presence of self-ligation products is detected through the second restriction enzyme digestion step. The adapter sequence acts as a marker that provides feedback about whether self-ligation occurred, allowing selective removal of self-ligated fragments while preserving properly ligated fragments that have different sequences at the second restriction site.
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 allows for precise and reliable analysis of genomic DNA methylation levels by distinguishing between self-ligation and hetero-ligation products, enabling consistent results across experiments and improving the accuracy of methylation percentage determination.
Implementation Method 1
digesting the twin adapter with the first RE, resulting in two adapters
Implementation Method 2
digesting a genomic DNA with at least a second restriction enzyme, wherein the digestion by the second RE results in the genomic DNA being broken into a plurality of restriction fragments
Implementation Method 3
ligating with a DNA ligase the adapter to the plurality of restriction fragments, resulting in a mixture of a self-ligation product and a hetero-ligation product
Data Source
AI summary
The disclosed methods in the application relates to making an adapter-tagged restriction library. The library or libraries created with the disclosed methods avoid the self-ligation of the restriction fragments. The application further disclosed methods of using the library or libraries to measure the methylation level of a genome, or comparing methylation levels among two or more genomes. The measurement of the global methylation levels can be achieved with quantitative PCR method by measuring the number of restriction fragments in the libraries. It also can be used for next generation Sequencing (NGS), Copy Number Variation (CNV), restriction site mutation, endogenous gene jumping, and exogenous DNA insertion, somatic hypermutation, gene knockout/knock in etc.