Direct ATP Release Sequencing via Tetraphosphate Moieties
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Solution Overview
Problem
Current pyrosequencing methods rely on the release of pyrophosphate (PPi) to initiate detectable light signals for nucleotide incorporation, which limits the sequencing process's efficiency and accuracy.
Innovation Solution
The use of adenosine-2′-deoxynucleoside tetraphosphate moieties in a reagent mix with a polymerase under primer extension conditions allows for direct detection of ATP production, which is proportional to nucleotide incorporation, enabling more precise and efficient sequencing without relying on PPi release for light signal initiation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If pyrosequencing relies on PPi release to initiate detectable light signals, then nucleotide incorporation can be detected, but sequencing efficiency and accuracy are limited
Solution Approach 1:
The invention extracts and directly detects the ATP molecule released during nucleotide incorporation, rather than relying on the indirect PPi detection pathway. By using an ATP-specific detector (such as an ATP-binding protein or fluorescent ATP probe), the method directly measures ATP production, which occurs stoichiometrically with nucleotide incorporation, thereby improving both sequencing efficiency and measurement precision
Solution Approach 2:
The invention introduces an ATP detection system as an intermediary between nucleotide incorporation and signal detection. This intermediary system (ATP detector) provides a more direct and sensitive transduction pathway compared to the traditional PPi-sulfurylase-luciferase cascade, enabling improved accuracy and efficiency in sequencing
2Reliability
If traditional pyrosequencing methods are used, then sequencing can be performed, but the process requires multiple enzymatic steps that reduce sensitivity and precision
Solution Approach 1:
The invention extracts and directly detects the ATP molecule released during nucleotide incorporation, rather than relying on the indirect PPi detection pathway. By using an ATP-specific detector (such as an ATP-binding protein or fluorescent ATP probe), the method directly measures ATP production, which occurs stoichiometrically with nucleotide incorporation, thereby improving both sequencing efficiency and measurement precision
Solution Approach 2:
Instead of following the traditional pathway where PPi is converted to ATP by sulfurylase and then detected via luciferase, the invention inverts the detection strategy by directly detecting ATP as it is released during polymerization. This reversal eliminates the need for the PPi-sulfurylase-luciferase cascade, reducing complexity while improving reliability
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 enhances sequencing efficiency by directly detecting ATP production, improving the accuracy of nucleotide identification and allowing for longer sequences to be determined with increased sensitivity and precision.
Implementation Method 1
The reagent mix may contain an adenosine-2'-deoxynucleoside tetraphosphate moiety and a polymerase
Implementation Method 2
Subsequently, this ATP is consumed in a luciferin-luciferase reaction to generate PPi and detectable light in amounts that are proportional to the amount of ATP
Data Source
AI summary
A method for sequencing a nucleic acid is provided. In certain embodiments, the method includes contacting a nucleic acid duplex comprising a nucleic acid template and a primer annealed to the template with a reagent mix under primer extension conditions to produce an extended primer and ATP. The reagent mix may contain an adenosine-2′-deoxynucleoside tetraphosphate moiety and a polymerase. The method further includes detecting the produced ATP. Also provided are adenosine-2′-deoxynucleoside tetraphosphate moieties that find use in the subject methods. Also provided are kits containing the adenosine-2′-deoxynucleoside tetraphosphate moieties for use in the subject methods.