Nucleic Acid Isolation via Oligonucleotide Tagging and PCR
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Solution Overview
Problem
Current methods for isolating target nucleic acid sequences from a population are slow, expensive, and inefficient, requiring overnight bacterial transformation and colony growth, with limitations in accuracy and throughput.
Innovation Solution
The method involves attaching oligonucleotide tags to nucleic acid molecules, amplifying them, and using microparticles with complementary sequences to isolate and amplify target nucleic acid molecules, enabling high-throughput sequencing and efficient isolation through techniques like limiting dilution and PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional bacterial transformation and colony growth methods are used, then target nucleic acid sequences can be isolated, but the process is slow and time-consuming
Solution Approach 1:
The patent replaces the biological bacterial transformation system with a chemical PCR amplification system. Instead of using bacteria to replicate and isolate nucleic acid sequences, the invention uses polymerase chain reaction with specific primers to directly amplify target sequences from the library in vitro, eliminating the need for bacterial culture and colony picking while achieving the same isolation goal
Solution Approach 2:
The patent creates multiple copies of the target nucleic acid sequence through PCR amplification. By using sequence-specific primers that bind to unique regions of the target molecule, the system generates numerous identical copies of the desired sequence from the original library, enabling detection and isolation without relying on single bacterial colonies
2Measurement precision
If traditional transformation methods are used, then target sequences can be identified, but the cost is high
Solution Approach 1:
The patent replaces expensive biological transformation procedures with cost-effective chemical PCR amplification. The in vitro amplification method uses readily available reagents and standard laboratory equipment, eliminating the need for expensive bacterial strains, transformation protocols, and colony screening procedures while maintaining high identification accuracy
Solution Approach 2:
The patent changes the fundamental parameters of the isolation process by shifting from biological replication (bacterial growth) to chemical replication (PCR amplification). This parameter change includes using thermal cycling instead of incubation, specific primer annealing temperatures, and enzymatic amplification instead of cellular division, resulting in reduced time and cost
3Manufacturing precision
If limiting dilution is used to isolate single cells, then single molecules can be obtained, but the throughput is low
Solution Approach 1:
The patent merges the functions of single-molecule isolation and amplification into a single PCR reaction. Instead of first isolating single molecules through limiting dilution and then separately amplifying them, the invention combines these steps by using PCR to amplify individual target sequences directly from the library mixture, achieving both precision and higher throughput
Solution Approach 2:
The patent transitions from a spatial separation approach (limiting dilution requiring physical separation of molecules into different wells) to a chemical amplification approach (PCR allowing simultaneous processing of multiple targets in parallel reactions). This dimensional change enables high-throughput isolation by processing many samples concurrently rather than sequentially
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 significantly increases the accuracy, yield, and cost efficiency of nucleic acid synthesis and assembly, allowing for precise isolation and identification of target sequences from large libraries, reducing the time and resources required.
Implementation Method 1
providing a plurality of microparticles, wherein each microparticle has an oligonucleotide sequence complementary to a portion of the nucleic acid molecules immobilized on its surface; forming a population of nucleic acid molecules hybridized to the complementary oligonucleotide sequence on the microparticles
Implementation Method 2
amplifying the plurality of nucleic acid-oligonucleotide tag molecules using primers complementary to the plurality of oligonucleotide tags
Data Source
AI summary
Methods and devices relate to the isolation of nucleic acids of interest from within a population of nucleic acids such as libraries of nucleic acid sequences.