Probe-Linked RNA Analysis With Gap Filling and Barcoding
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Solution Overview
Problem
Current methods for analyzing RNA, such as PCR and sequencing, face challenges in efficiently and accurately processing biological samples within partitions, particularly in filling gaps between probe ends hybridized to target sequences, which can affect the accuracy and efficiency of sequence identification.
Innovation Solution
The use of Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) or prokaryotic Y-family DNA polymerases to fill the gap regions between probe ends hybridized to target RNA sequences, followed by ligation to generate extended probes, which can include barcode sequences for sample or cell identification, and subsequent reverse transcription to generate cDNA molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional PCR and sequencing methods are used for RNA analysis, then the process is straightforward, but the accuracy and efficiency of processing biological samples within partitions is reduced
Solution Approach 1:
The method divides the RNA analysis process into distinct partitioned reactions, where individual RNA molecules or pools of molecules are processed in separate micro-compartments. This segmentation enables parallel processing of multiple samples simultaneously, improving throughput while maintaining accurate sequence identification through isolated probe hybridization and gap-filling reactions in each partition.
Solution Approach 2:
The protocol performs preliminary probe hybridization to target RNA sequences before the gap-filling step. Probes are designed with 5' and 3' ends that hybridize to known flanking regions of the target RNA,预先 establishing the framework for accurate gap filling. This preliminary action ensures that subsequent polymerase-mediated gap filling occurs at precisely defined locations, enhancing both accuracy and efficiency.
2Measurement precision
If gap regions between probe ends are not filled, then the process is simpler, but the accuracy of sequence identification is reduced
Solution Approach 1:
The method employs a specialized DNA polymerase (such as Phusion or Q5 polymerase) as an intermediary enzyme to fill the gap regions between hybridized probe ends. This intermediary enzyme mediates the incorporation of nucleotides complementary to the target RNA sequence in the gap region, enabling accurate sequence reconstruction without requiring direct sequencing of the gap itself. The polymerase acts as a bridge that connects the known probe sequences to the unknown target sequence.
Solution Approach 2:
The protocol optimizes reaction parameters for the gap-filling step, including adjusting magnesium ion concentration, temperature, and polymerase concentration to enhance the fidelity and efficiency of gap filling. By carefully controlling these parameters, the method achieves high accuracy in sequence identification while managing the complexity of the gap-filling process through standardized reaction conditions.
3Adaptability or versatility
If multiple probes are used to cover different target sequences, then the versatility of analysis is improved, but the complexity of probe design and processing increases
Solution Approach 1:
The method employs universal probe design elements and standardized gap-filling protocols that can be applied across different target RNA sequences. The probe structure incorporates universal flanking regions that hybridize to conserved sequences, allowing the same gap-filling and sequencing approach to be used for diverse targets. This universality enables the system to analyze different target sequences without requiring fundamentally different methodologies, managing complexity through standardization while maintaining versatility.
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
Enhances the accuracy and efficiency of RNA analysis by enabling precise filling of gaps between probe ends, allowing for effective sequence identification and sample or cell-specific barcoding, thereby improving the quality and quantity of nucleic acid processing.
Implementation Method 1
filling the gap region using a Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), thereby generating an extended first probe end
Implementation Method 2
a first probe end hybridized to the first target sequence, and a second probe end hybridized to the second target sequence
Implementation Method 3
generating a probe-linked nucleic acid molecule comprising the at least one probe and the gap region
Data Source
AI summary
Provided herein are systems and methods for analyzing biomolecules (e.g., nucleic acid molecules, proteins). A method of analyzing a target ribonucleic acid (RNA), can include: (a) providing: (i) a target RNA, comprising a first target sequence and a second target sequence, and (ii) at least one probe including a first probe end hybridized to the first target sequence, and a second probe end hybridized to the second target sequence, wherein the first probe end hybridized to the first target sequence and the second probe end hybridized to the second target sequence are separated by a gap region. The method can further include filling the gap region and generating a probe-linked nucleic acid molecule including the at least one probe and the gap region.