Thermodynamic Parameter Calculation for Nucleic Acid Hybridization
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Solution Overview
Problem
Existing methods for predicting thermodynamic parameters of oligonucleotides, particularly for multiplex PCR, are inaccurate due to errors in primer design and require empirical testing, leading to inefficiencies and inconsistencies.
Innovation Solution
A computer-implemented system and method for calculating the change in enthalpy (ΔH°) and the change in entropy (ΔS°) for the melting of individual oligonucleotides from experimental data, using advanced data analysis techniques such as singular value decomposition and global fitting to minimize errors and optimize results.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If existing computer programs are used to pick and design PCR primers, then primer design can be automated, but errors in prediction lead to additive errors in multiplex PCR requiring empirical testing
Solution Approach 1:
The patent changes the thermodynamic parameters used for prediction from traditional nearest-neighbor models to a novel set of parameters derived from melting temperature measurements at multiple concentrations. This parameter transformation enables more accurate prediction of hybridization strength, reducing errors in automated primer design while maintaining automation benefits
Solution Approach 2:
The patent replaces empirical trial-and-error mechanical testing with a computational thermodynamic model. By substituting physical empirical testing with a mathematical framework based on thermodynamic principles and multiple concentration measurements, the system achieves higher reliability in predicting primer performance in multiplex PCR
2Ease of manufacture
If traditional thermodynamic parameters are used for predicting hybridization strength, then calculations are simple, but accuracy is insufficient for oligonucleotides with multiple nucleic chemistries
Solution Approach 1:
The patent transforms the thermodynamic parameter set from traditional nearest-neighbor parameters to a new parameter system based on melting temperature measurements at multiple concentrations. This parameter change enables accurate prediction of hybridization strength for complex oligonucleotides containing multiple chemistries (DNA, RNA, LNA, PNA) while maintaining computational tractability through a systematic calculation framework
Solution Approach 2:
The patent develops a unified thermodynamic model that treats hybridization of composite oligonucleotides (containing multiple nucleic acid chemistries) as a single system. By creating composite thermodynamic parameters that account for interactions between different chemistries, the model achieves accurate predictions for hybrid molecules without requiring separate calculations for each chemistry type
3Reliability
If empirical testing is performed to validate primer performance, then accuracy can be improved, but time consumption and throughput are reduced
Solution Approach 1:
The patent performs preliminary thermodynamic calculations using the novel multi-concentration melting temperature method to predict primer performance before actual PCR experiments. This preliminary action provides highly accurate predictions that reduce or eliminate the need for extensive empirical testing, thereby maintaining high throughput while ensuring reliability
Solution Approach 2:
The patent creates a computational model that copies and simulates the physical hybridization process through thermodynamic calculations. By using mathematical models that replicate the behavior of actual PCR reactions, the system can predict primer performance in silico, reducing the need for physical empirical testing and increasing design throughput
Data Source
AI summary
A dilution of an oligonucleotide duplex is made. One aliquot of the dilution is added to a double-stranded nucleic acid (dsNA) melt at low concentration. A second aliquot of the dilution is added to a dsNA melt at high concentration. A third aliquot of the dilution is added to a reference cuvette containing only a first strand of the duplex. A fourth aliquot of the dilution is added to a reference cuvette containing only a second strand of the duplex. A first dsNA melt curve, a second dsNA melt curve, a first strand absorbance versus temperature curve, and a second strand absorbance versus temperature curve are produced. ΔH° and ΔS° are calculated for the first strand and the second strand from a fit to the first dsNA melt curve, the second dsNA melt curve, the first strand absorbance versus temperature curve, and the second strand absorbance versus temperature curve.