Nucleic Acid Normalization via Limiting Endonuclease Cleavage
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Solution Overview
Problem
Conventional methods for normalizing nucleic acid samples are time-consuming, labor-intensive, prone to human error, and often require sacrificing samples or specialized equipment, limiting their automation in high-throughput laboratories.
Innovation Solution
The method involves reacting samples with a limiting amount of a single-turnover sequence-specific endonuclease that recognizes a target sequence, cleaving a portion of the nucleic acid molecules, and isolating or amplifying the resulting cleavage product, allowing for proportional adjustment of nucleic acid amounts without quantification, enabling normalization of specific sequences within or between samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional normalization methods are used, then nucleic acid sample concentration can be measured and adjusted, but the process becomes time-consuming and labor-intensive
Solution Approach 1:
The patent extracts the normalization function from manual measurement and calculation processes, embedding it directly into the PCR amplification step through limiting reagents. This eliminates the need for separate quantification steps while maintaining normalization accuracy.
Solution Approach 2:
The patent performs normalization preparation in advance by designing primers or probes with built-in normalization capabilities. The limiting reagent approach pre-establishes the normalization framework before the actual measurement and adjustment steps would normally occur.
2Measurement precision
If conventional normalization methods are used, then nucleic acid concentration can be adjusted, but the process requires specialized equipment and sample sacrifice
Solution Approach 1:
The patent makes the normalization process universal by integrating it into standard PCR workflows. The same primers and reaction conditions used for amplification also perform normalization, eliminating the need for specialized equipment while maintaining quantification accuracy.
3Ease of operation
If manual normalization procedures are used, then concentration adjustment can be performed, but human error increases and automation becomes limited
Solution Approach 1:
The patent implements self-service normalization where the reaction system automatically performs concentration normalization through the limiting reagent mechanism. The system self-regulates without human intervention, eliminating manual errors while maintaining ease of operation through simple protocol addition.
4Measurement precision
If quantification steps are performed before normalization, then accurate measurement is achieved, but the process becomes more complex and time-consuming
Solution Approach 1:
The patent merges the quantification and normalization steps into a single integrated process. The limiting reagent approach allows both measurement and concentration adjustment to occur simultaneously during the PCR reaction, doubling productivity while maintaining measurement precision.
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 significantly reduces the need for quantification steps, minimizes sample loss, and can be automated, providing accurate and efficient normalization of nucleic acid samples for further analysis, such as sequencing, while enriching for specific sequences.
Implementation Method 1
a single-turnover sequence-specific endonuclease that recognizes a target sequence
Implementation Method 2
cleaving a portion of the nucleic acid molecules that comprise the target sequence and producing a normalized amount of a first cleavage product
Data Source
Figure 1
AI summary
Among other things, a method for normalizing a sample is provided. In some embodiments, the method comprises: (a) reacting a sample with a limiting amount of a single-turnover sequence-specific endonuclease that recognizes a target sequence, thereby cleaving a portion of the nucleic acid molecules that comprise the target sequence and producing a normalized amount of a first cleavage product; and (b) isolating, transcribing or selectively amplifying the normalized amount of the first cleavage product. In this method, because a limiting amount of the endonuclease is used, the normalized amount of the first cleavage product is determined by the limiting amount of the first single-turnover sequence-specific endonuclease used in step (a).