Nucleic Acid Purification via Primer Hybridization and Selective NTP Incorporation
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Solution Overview
Problem
Current nucleic acid purification methods, particularly for RNA, have a low efficiency of less than 80% and are unable to effectively separate normal RNA from RNA with one-base errors, which are common during synthesis.
Innovation Solution
A method involving hybridization of a nucleic acid library with a complementary primer, followed by reaction with nucleotide triphosphates containing irreversible or blocker-free blockers, such as ddNTPs or Azido-dNTPs, to selectively bind and isolate target nucleic acids based on base specificity, using multiplex oligonucleotide library purification by synthesis and selection (MOPSS) on magnetic beads.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If current universal purification methods are used, then the purification process is simple and quick, but the purification rate is less than 80% and cannot separate RNA with one-base error
Solution Approach 1:
The patent applies local quality by designing primers with specific base sequences that complement target RNA molecules. The primers are designed to bind specifically to sequences of interest, allowing selective purification of RNA molecules with particular sequences while leaving others unchanged. This enables differentiation between correct and erroneous RNA sequences at the local sequence level.
Solution Approach 2:
The patent utilizes parameter changes by employing nucleotide triphosphates with modified properties (such as irreversible blockers or blocker-free variants) that alter the synthesis process. These modified NTPs change the chemical parameters of the reaction to enable selective incorporation only at specific positions, thereby achieving high-precision purification based on sequence parameters.
2Measurement precision
If conventional purification methods are used, then the process is straightforward, but it cannot distinguish normal RNA from RNA with one-base error
Solution Approach 1:
The patent introduces an intermediary mechanism using primers and nucleotide triphosphates as mediators between the target RNA and the detection/purification system. The primers bind to the RNA and serve as intermediaries that guide the selective incorporation of NTPs, enabling precise detection and purification of RNA molecules with specific sequences or errors.
Solution Approach 2:
The patent replaces conventional mechanical purification methods with a biochemical mechanism based on complementary base pairing and selective nucleotide incorporation. This substitution allows for molecular-level discrimination between correct and erroneous RNA sequences through chemical specificity rather than physical separation alone.
3Productivity
If high-purity purification is achieved through selective synthesis and selection, then the purification rate increases beyond 80%, but the method complexity increases
Solution Approach 1:
The patent applies preliminary action by performing selective synthesis and selection steps before final purification. The process includes preliminary hybridization of primers with target RNA, followed by selective incorporation of NTPs, and then separation based on the resulting differences. This staged approach enables high purification efficiency by eliminating errors early in the process.
Solution Approach 2:
The patent incorporates feedback mechanisms where the incorporation of NTPs with irreversible blockers or blocker-free variants provides information about the sequence accuracy. The selective binding and subsequent separation steps use this feedback to identify and isolate only the correct RNA molecules, continuously refining the purification based on sequence verification.
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 allows for high-purity purification and analysis of target nucleic acids, specifically distinguishing and isolating RNA with one-base errors, thereby improving the yield and accuracy of RNA purification beyond existing methods.
Implementation Method 1
hybridizing a library containing the target nucleic acid with a primer that binds complementarily to the target nucleic acid
Implementation Method 2
a blocker-free NTP selected from the group consisting of blocker-free ATP, CTP, GTP, and TTP that binds to the target nucleic acid
Data Source
AI summary
The present invention relates to a method for purifying a nucleic acid and a method for analyzing the presence of a target nucleic acid in a sample, and relates to a method for purifying a target nucleic acid from a nucleic acid library to high purity or a method for analyzing the presence of a target nucleic acid in a sample. Specifically, the present invention relates to a method for purifying and analyzing a nucleic acid for selecting a target nucleic acid based on whether a nucleic acid complementary to the target nucleic acid is synthesized.