Fluorinated Deoxyribonucleoside Polyphosphates for PCR Stability
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Nucleic acid amplification reagents, particularly deoxyribonucleoside triphosphates, are temperature sensitive and prone to degradation, leading to the formation of nucleoside diphosphates and monophosphates that are no longer substrates for nucleic acid polymerases, and their stability can be compromised by esterification which affects enzyme activity.
Innovation Solution
The use of fluorinated deoxyribonucleoside polyphosphates as modified nucleotide reagents that are thermally stable and can withstand high temperatures, maintaining their substrate efficiency for polymerase enzymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If deoxyribonucleoside triphosphates are used as nucleic acid amplification reagents, then they can serve as substrates for nucleic acid polymerases, but they are prone to degradation via loss of phosphate groups leading to formation of inactive nucleoside diphosphates and monophosphates
Solution Approach 1:
The patent applies parameter changes by modifying the terminal phosphate group of deoxyribonucleoside triphosphates through esterification with various alcohols (methanol, ethanol, propanol, butanol). This chemical modification changes the stability parameter of the nucleotide reagent, preventing spontaneous degradation while maintaining polymerase substrate activity. The esterified terminal phosphate groups remain stable under heat stress conditions that completely degrade normal dNTPs.
2Reliability
If the terminal phosphate is esterified to improve thermal stability, then the nucleotide becomes resistant to degradation, but the ability to serve as an effective substrate for certain polymerase enzymes is reduced
Solution Approach 1:
The patent systematically varies the esterification parameter by using different alcohol chains (methanol, ethanol, propanol, butanol) to create a series of dNTP analogs with progressively longer alkyl groups. This gradient approach allows optimization of the balance between thermal stability and polymerase substrate efficiency. The data shows that while all esterified analogs improve stability, the degree of polymerase compatibility varies with the specific ester group used.
Solution Approach 2:
The patent applies local quality by selectively modifying only the terminal phosphate group while leaving the rest of the nucleotide structure (base, sugar, and inner phosphate groups) unchanged. This localized modification approach preserves the essential polymerase recognition elements while conferring thermal stability through the esterified terminal phosphate, thus resolving the contradiction between stability and enzyme compatibility.
3Ease of manufacture
If nucleic acid amplification reagents are stored at ambient temperature, then storage and shipping costs are reduced, but temperature sensitive components degrade and lose activity
Solution Approach 1:
The patent fundamentally changes the temperature parameter requirement for storage by modifying the chemical structure of the nucleotide reagents through esterification. This structural modification shifts the stability parameter such that the reagents can now be stored at ambient temperature (20-25°C) without degradation, eliminating the need for expensive cold chain storage and shipping while maintaining full reagent activity.
Data Source
AI summary
The present invention provides for stable nucleotide reagents used for nucleic acid amplification by PCR and RT-PCR (Reverse Transcriptase-PCR) that comprises modified nucleoside polyphosphates. The present invention also provides for methods for using the modified nucleoside polyphosphates for detecting the presence or absence of a target nucleic acid sequence in a sample in an amplification reaction.