Single-Stranded Nucleic Acid Probes for High-Throughput Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for generating oligonucleotide libraries are costly and labor-intensive, making it challenging to achieve high-throughput sequencing efficiently, particularly in pharmacogenomics where comprehensive genetic analysis is required for understanding diseases and drug response.
Innovation Solution
A method involving the generation of single-stranded nucleic acid probes with predetermined sequences through a process of restriction enzyme manipulation and amplification, allowing for the creation of high-quality probe populations for use in high-throughput sequencing platforms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If serial analysis of individual DNA samples is performed, then analysis accuracy is improved, but cost and time requirements increase significantly
Solution Approach 1:
The method segments the analysis process by creating pooled DNA samples from multiple individuals, where each pool is analyzed together rather than individually. This allows parallel processing of multiple samples through a single sequencing run, maintaining analytical accuracy while dramatically improving throughput and reducing costs.
Solution Approach 2:
The invention merges multiple individual DNA samples into pooled samples that can be analyzed simultaneously. By combining samples from multiple individuals into pools and using pool-specific barcodes, the method enables concurrent analysis of multiple individuals in a single sequencing experiment, resolving the contradiction between accuracy and productivity.
2Manufacturing precision
If oligonucleotide libraries are generated using conventional methods, then probe quality is maintained, but generation cost and time increase
Solution Approach 1:
The method employs preliminary action by pre-synthesizing oligonucleotide probes with universal adapter sequences and restriction sites before the actual sequencing experiment. These pre-prepared probes are then amplified and pooled, allowing rapid generation of high-quality probe libraries without time-consuming conventional synthesis methods for each individual sample.
Solution Approach 2:
The invention uses copying by amplifying a small set of universal probe sequences through PCR to generate large populations of identical probes. This exponential amplification allows conversion of a small initial probe set into sufficient quantities for high-throughput sequencing, dramatically reducing generation time while maintaining probe quality through controlled amplification.
3Productivity
If high-throughput sequencing is implemented, then productivity is improved, but the complexity of sample processing increases
Solution Approach 1:
The method applies universality by designing a single universal probe sequence with universal adapter sequences that can bind to multiple different DNA samples. This universal probe design simplifies processing complexity while enabling high-throughput sequencing of diverse samples, as the same probe protocol works across all pool types without requiring sample-specific probe synthesis.
Solution Approach 2:
The invention introduces intermediary elements in the form of universal adapter sequences and restriction sites that mediate between the sequencing platform and diverse DNA samples. These intermediaries standardize the interaction interface, allowing complex sample processing to be simplified through uniform binding and amplification protocols while maintaining high throughput capability.
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 reduces the cost and time required for generating oligonucleotide libraries, enabling efficient and comprehensive genetic analysis by providing a cost-effective method for producing high-quality probes for sequencing and capture applications.
Implementation Method 1
contacting the 5' and 3' flanking regions of the linear double-stranded nucleic acid precursor molecules with the first and second restriction enzymes to cleave the first and second restriction enzyme recognition sequences so as to generate the ligation substrates
Implementation Method 2
ligating the ligation substrates together so as to generate a plurality of random head-to-tail concatemers
Implementation Method 3
contacting the released precursor molecules of step (e) above with alkaline phosphatase
Implementation Method 4
contacting the released precursor molecules of step (e) above with an exonuclease so as to selectively degrade the one strand of the double-stranded monomer linear precursor molecules
Implementation Method 5
the single-stranded nucleic acid probes further comprise a region which hybridizes to a capture nucleic acid molecule
Data Source
AI summary
Provided herein are compositions and kits for single-stranded nucleic acid probes, and methods for making the single-stranded nucleic acid probes, where the single-stranded nucleic acid probes comprise a probe region having a predetermined sequence which is flanked by a 5′ region having a first restriction enzyme recognition sequence and flanked by a 3′ region having a second restriction enzyme recognition sequence, and a region which hybridizes to a capture nucleic acid molecule. The single-stranded nucleic acid probes are useful for solution-based capture methods.