Second Strand cDNA Synthesis via Self-Priming Tagmentation
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Solution Overview
Problem
Current methods for generating cDNA libraries compatible with high-throughput sequencing platforms suffer from low yields, lack of reproducibility, and high costs.
Innovation Solution
A method involving the synthesis of a double-stranded mRNA:cDNA hybrid, followed by second strand cDNA synthesis using an enzyme with RNase H activity, and subsequent tagging with adapter sequences using an adapter-loaded tagmentase, to produce sequencing platform-specific cDNA amplicons.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard in vitro methods are used for constructing cDNA libraries with adaptors, then library construction can be performed, but yields are low and costs are high
Solution Approach 1:
The patent employs self-priming mechanisms where the adapter sequences themselves serve as primers for DNA synthesis. The 5' adapter on the first strand cDNA and the 3' adapter on the second strand cDNA automatically prime the synthesis reactions without requiring additional external primers, thereby simplifying the protocol and improving yield while reducing reagent costs
Solution Approach 2:
The patent performs preliminary adapter tagging during the cDNA synthesis process itself, rather than as a separate subsequent step. The adapters are incorporated into the cDNA strands during reverse transcription and second-strand synthesis, enabling downstream amplification and sequencing without requiring additional ligation or tagging steps
2Reliability
If standard in vitro methods are used for constructing cDNA libraries with adaptors, then library construction can be performed, but reproducibility is poor
Solution Approach 1:
The patent merges multiple separate steps into a unified workflow. The adapter tagging, second-strand synthesis, and amplification priming functions are combined into an integrated process where the same adapter sequences serve multiple purposes: as tags for identification, as primers for synthesis, and as binding sites for amplification. This consolidation reduces protocol complexity and improves reproducibility
Solution Approach 2:
The adapter sequences designed in this patent are universal and multi-functional. They can be used across different cDNA library construction scenarios and are compatible with various amplification methods. The same adapter structure serves as a primer binding site, a tag for sequencing identification, and a handle for enzymatic reactions, eliminating the need for multiple specialized reagents
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 method enhances the efficiency of cDNA synthesis, increases reproducibility, and reduces costs by improving the yield and quality of cDNA libraries for high-throughput sequencing.
Implementation Method 1
contacting the mRNA:cDNA hybrid with an enzyme comprising RNase H activity, thereby producing mRNA fragments hybridized to the first strand cDNA
Implementation Method 2
contacting the mRNA fragments with a DNA polymerase, thereby extending the mRNA fragments in a template-directed polymerase reaction
Implementation Method 3
contacting the double-stranded cDNA polynucleotide with an adapter-loaded tagmentase, thereby forming a reaction mixture comprising a tagged double-stranded cDNA polynucleotide
Data Source
AI summary
Methods and compositions are provided herein for preparing high-throughput cDNA sequencing libraries.