Oligonucleotide Synthesis Efficiency Estimation via LC-MS
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Solution Overview
Problem
Existing methods for synthetic oligonucleotide synthesis struggle to accurately measure single base deletion rates, leading to potential frame shifts and non-functional genes, as they lack efficient means to assess the quality and efficiency of oligonucleotide synthesis reactions.
Innovation Solution
A method involving liquid chromatography-mass spectrometry (LC-MS) to analyze mass spectra, estimate the efficiency of oligonucleotide synthesis reactions, and quantify single base deletion rates for individual nucleotides (G, A, T, C) without requiring DNA sequencing, providing rapid and accurate results.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional DNA sequencing methods are used to measure single base deletion rates, then measurement accuracy is improved, but analysis time and complexity increase significantly
Solution Approach 1:
The patent extracts and measures only the specific n-1 reaction products (oligonucleotides missing a single base) from the complex synthesis mixture using LC-MS, rather than performing complete DNA sequencing. This selective extraction of the problematic component enables rapid quantification of deletion rates without the time-consuming nature of full sequencing analysis
Solution Approach 2:
The patent replaces the mechanical/enzymatic DNA sequencing process with a chemical analysis approach using liquid chromatography-mass spectrometry. This substitution measures the mass-to-charge ratio of oligonucleotide fragments to identify and quantify deletion products, achieving rapid results without the complex biological machinery required for traditional sequencing
2Manufacturing precision
If comprehensive quality analysis of oligonucleotide synthesis is performed, then manufacturing precision is improved, but device complexity and analysis difficulty increase
Solution Approach 1:
The patent employs LC-MS technology that serves multiple functions: it separates oligonucleotide fragments by size, identifies them by mass-to-charge ratio, and quantifies their abundance. This single analytical platform provides comprehensive quality assessment including overall cycle yield and individual nucleotide deletion rates, replacing multiple specialized analytical methods
Solution Approach 2:
The patent segments the quality analysis into distinct measurable components: full-length oligonucleotides, n-1 reaction products (single base deletions), and potentially other incomplete products. By analyzing each segment separately through LC-MS, the method achieves comprehensive quality control while maintaining analytical simplicity through systematic breakdown of the synthesis outcome
3Productivity
If rapid quality assessment methods are used for oligonucleotide synthesis, then productivity is improved, but measurement precision deteriorates
Solution Approach 1:
The patent performs preliminary separation and identification of oligonucleotide products using LC-MS before quantification. The mass spectrometer detects and characterizes n-1 reaction products based on their unique mass-to-charge ratios, allowing accurate identification and measurement of single base deletions to occur before final data analysis, ensuring both speed and 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
Enables precise estimation of overall and individual single base deletion rates, improving the quality control of oligonucleotide synthesis by determining cycle yields and deletion rates, thus enhancing the production of functional genes.
Implementation Method 1
subjecting the products of one or more oligonucleotide synthesis reactions to LC-MS
Implementation Method 2
subjecting the products of one or more oligonucleotide synthesis reactions to LC-MS to produce a series of mass spectra
Data Source
AI summary
Described herein, among other things, is a method of estimating efficiency of an oligonucleotide synthesis reaction. In some embodiments, the method comprises subjecting the products of one or more oligonucleotide synthesis reactions to LC-MS to produce a series of mass spectra, analyzing the mass spectra, and estimating the overall efficiency of an oligonucleotide synthesis reaction and/or the efficiency of addition of one or more of G, A, T or C individually in an oligonucleotide synthesis reaction.