Labeled Nucleotide Purity via Enzymatic Pre-treatment
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Solution Overview
Problem
Conventional methods for labeling and purifying nucleotides for nucleic acid analyses result in impure compositions, leading to high background noise and errors in sequencing and hybridization reactions due to the presence of unlabeled nucleotides, which are not fully incorporated by polymerase enzymes.
Innovation Solution
A composition of labeled nucleotides or nucleic acid targets substantially free of unlabeled counterparts is achieved by treating the mixture with agents like phosphatases or exonucleases, rendering unlabeled nucleotides incapable of participating in analytical reactions, thereby ensuring high-resolution detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional labeling and purification methods are used, then the process is simple and cost-effective, but the nucleotide composition contains unlabeled nucleotides leading to high background noise and errors
Solution Approach 1:
The patent applies preliminary action by treating the nucleotide mixture with phosphatase or exonuclease enzymes before the sequencing or hybridization reaction. This pre-treatment selectively degrades unlabeled nucleotides while leaving labeled nucleotides intact, thereby improving purity before the main analytical reaction occurs. The enzyme treatment step is performed in advance to prevent the harmful effects of unlabeled nucleotides during subsequent high-resolution detection.
2Productivity
If polymerase enzymes are used in nucleic acid analyses, then DNA synthesis can occur, but the enzymes prefer natural nucleotides over labeled nucleotide analogs resulting in missed bases
Solution Approach 1:
The patent applies preliminary anti-action by using phosphatase or exonuclease enzymes to selectively remove or modify unlabeled nucleotides before they can interfere with the polymerase reaction. This counter-action occurs in advance to prevent the polymerase from preferentially incorporating unlabeled nucleotides over labeled analogs, thereby eliminating the source of missed bases and improving measurement precision while maintaining productivity.
3Measurement precision
If high-resolution detection methods are used, then accurate detection of labeled nucleotides is achieved, but the presence of unlabeled nucleotides creates high background noise
Solution Approach 1:
The patent applies the extraction principle by using phosphatase or exonuclease enzymes to selectively remove unlabeled nucleotides from the mixture before high-resolution detection. This separation step extracts the harmful unlabeled components while retaining the labeled nucleotides, thereby eliminating background noise and enabling accurate high-resolution detection without interference from unlabeled contaminants.
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
The solution significantly reduces the presence of unlabeled nucleotides to less than 1%, minimizing errors and background noise in nucleic acid sequencing and hybridization assays, enhancing the accuracy of nucleic acid analyses.
Implementation Method 1
treating the mixture with an agent that selectively renders unlabeled nucleotides or nucleotide analogs substantially incapable of participating in an analytical reaction of interest... The agent can comprise an enzyme. In some embodiments, the enzyme is selected from the group consisting of a phosphatase
Implementation Method 2
treating the nucleic acid target composition with an agent that selectively renders unlabeled nucleic acid target substantially incapable of participating in a analytical reaction of interest... The agent is an exonuclease enzyme that selectively digests the unlabeled nucleic acid target
Implementation Method 3
a polymerase enzyme and divalent metal ion such as Mg2+ or Mn2+. detecting incorporation of the labeled nucleotide or nucleotide analog by the polymerase, into a nascent nucleic acid strand
Data Source
AI summary
The present invention relates to preparation of nucleotide compositions and uses thereof for conducting nucleic acid analyzes. The compositions and methods embodied in the present invention are particularly useful for nucleic acid analyzes that require high-resolution detection of labeled nucleotides or labeled nucleic acid targets.