Reversible Termination of Primer Extension Using 3'-ONH2 Nucleotides
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Solution Overview
Problem
Current methods for analyzing and manipulating nucleic acids using nucleoside triphosphate analogs face challenges in reversibly terminating primer extension, as enzymes like DNA polymerases, RNA polymerases, and reverse transcriptases struggle to accept and process analogs with a 3′-ONH2 group without causing damage or incomplete incorporation, leading to issues in sequencing and SNP detection.
Innovation Solution
Development of processes and compositions that utilize enzymes like TdT and Taq polymerase variants to incorporate and regenerate 3′-ONH2 terminated nucleotides, allowing for reversible termination and subsequent enzymatic extension, using buffered sodium nitrite and nitrous acid to regenerate the 3′-OH group, and employing Taq polymerase mutants with specific mutations to enhance the acceptance of these analogs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA polymerases or RNA polymerases are used to incorporate nucleoside triphosphate analogs with 3′-ONH2 group, then primer extension can be terminated, but the enzymes struggle to accept and process these analogs without causing damage or incomplete incorporation
Solution Approach 1:
The patent modifies the 3′-hydroxyl group of nucleoside triphosphate analogs by replacing it with a 3′-ONH2 group, creating a reversible terminator that can be incorporated by polymerases. This parameter change allows the analog to be accepted by the enzyme while providing controllable termination capability.
Solution Approach 2:
The 3′-ONH2 group serves as an intermediary blocking group that temporarily prevents further extension but can be chemically converted to restore the 3′-OH group. This intermediary approach enables reversible termination without permanently damaging the oligonucleotide.
2Reliability
If 3′-ONH2 group is used to block primer extension, then reversible termination is achieved, but the group must be removed to regenerate 3′-OH for further extension
Solution Approach 1:
The patent converts the potentially harmful effect of the 3′-ONH2 group (blocking further extension) into a beneficial reversible termination mechanism. By using chemical treatment to convert 3′-ONH2 back to 3′-OH, the blocking group becomes a controllable terminator rather than a permanent obstacle.
Solution Approach 2:
The sequencing process uses periodic cycles of incorporation and cleavage. During each cycle, the 3′-ONH2 group is incorporated to terminate extension, then subsequently cleaved to regenerate 3′-OH for the next cycle, creating a rhythmic pattern of termination and restoration.
3Productivity
If buffered sodium nitrite and nitrous acid are used to regenerate 3′-OH group, then high-yield regeneration is achieved, but the oligonucleotide must survive the washing, detecting and cleaving processes
Solution Approach 1:
The patent uses buffered sodium nitrite and nitrous acid to chemically convert the 3′-ONH2 group back to 3′-OH through controlled parameter changes in pH and reagent concentration. This chemical transformation achieves high-yield regeneration while minimizing damage to the oligonucleotide.
4Productivity
If Taq polymerase mutants with specific mutations are employed to enhance acceptance of analogs, then incorporation efficiency is improved, but the enzyme specificity may be reduced
Solution Approach 1:
The patent introduces specific mutations in Taq polymerase (such as A710T, A710V, A710I) to change the enzyme's substrate acceptance parameters. These mutations enhance the enzyme's ability to incorporate the modified nucleoside triphosphate analogs with 3′-ONH2 groups while maintaining sufficient specificity for accurate sequencing.
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 high-yield regeneration of 3′-OH groups in oligonucleotides, allowing for faithful and efficient enzymatic processing, including PCR amplification and sequencing, while maintaining the integrity of the oligonucleotide, and facilitates the detection of single nucleotide polymorphisms by competing reversible and irreversible terminators.
Implementation Method 1
buffered sodium nitrite and nitrous acid to regenerate the 3′-OH group
Implementation Method 2
enzymes that synthesize oligonucleotides from triphosphates, including terminal transferases, RNA polymerases, reverse transcriptases, and DNA polymerases
Data Source
AI summary
Processes are disclosed that use 3′-reversibly terminated nucleoside triphosphates to analyze DNA for purposes other than sequencing using cyclic reversible termination. These processes are based on the unexpected ability of terminal transferase to accept these triphosphates as substrates, the unexpected ability of polymerases to add reversibly and irreversibly terminated triphosphates in competition with each other, the development of cleavage conditions to remove the terminating group rapidly, in high yield, and without substantial damage to the terminated oligonucleotide product, and the ability of reversibly terminated primer extension products to capture groups. The presently preferred embodiments of the disclosed processes use a triphosphate having its 3′-OH group blocked as a 3′-ONH2 group, which can be removed in buffered NaNO2 and use variants of Taq DNA polymerase, including one that has a replacement (L616A).