Labeled Nucleotide C-Linker Reduces Background Peaks
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Solution Overview
Problem
Current nucleic acid amplification and detection methods, such as capillary electrophoresis, suffer from background peaks due to artefacts like degraded products or primers, leading to reduced sensitivity in DNA analysis.
Innovation Solution
The use of oligonucleotides with labeled nucleotides attached via a C-linker comprising a negative charged residue, specifically with a formula that includes a phosphate group and a fluorophore, reduces background peaks and enhances sensitivity by stabilizing the amplification products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescent labeling is used in nucleic acid amplification, then detection is enabled, but background peaks appear due to free labels and short oligonucleotides reducing sensitivity
Solution Approach 1:
The invention extracts the problematic free fluorescent labels and short oligonucleotides from the system by designing a linker that prevents their formation and release. The C-linker with negative charged residue ensures the label remains covalently bound to the oligonucleotide throughout the amplification process, eliminating the source of background peaks while maintaining detection capability.
Solution Approach 2:
The invention changes the chemical parameters of the linker by introducing a C-linker with a negative charged residue (such as a carboxylate or phosphate group). This parameter change increases the stability of the label-oligonucleotide conjugate and prevents degradation that would otherwise release free labels causing background peaks.
2Reliability
If oligonucleotides are stabilized to reduce background peaks, then detection sensitivity increases, but linker structure complexity increases
Solution Approach 1:
The invention applies local quality by introducing a specific functional feature (negative charged residue) at a specific location within the linker structure. Rather than complicating the entire linker, the solution focuses on adding the charge characteristic at the critical position where it provides maximum stability benefit with minimal structural complexity.
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 background noise and enhances the stability of oligonucleotides and amplification products, improving the sensitivity of nucleic acid detection and analysis.
Implementation Method 1
the use of an oligonucleotide as a primer, wherein said oligonucleotide comprises a nucleotide attached to a label via a C-linker comprising a negative charged residue
Data Source
Figure 1A~2
AI summary
The present application discloses a labeled nucleotide comprising a label attached via a linker, wherein said labeled nucleotide has the formula wherein R1 is a residue with a negative net charge, preferably selected from the group consisting of a phosphate group, and a sulphate group; wherein R2, R3 and R4 are independently selected from the group consisting of H2, OH2, and O; wherein "n" is an integer between 0 and 16; wherein "a" is an integer between 1 and 10; wherein SP is absent or a spacer; wherein X is said label; and wherein Y is a nucleotide or nucleoside. Furthermore, oligonucleotides comprising a labeled nucleotide according to the present invention and the use as a primer in amplification based methods is disclosed herein.