Ligation-Free Nucleic Acid Library Preparation for Degraded Samples
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Solution Overview
Problem
Conventional methods for preparing nucleic acid libraries for high-throughput sequencing, such as ligation-based techniques, are not suitable for small and highly degraded samples, and they can introduce untemplated nucleotides during in vitro transcription of RNAs, affecting their function.
Innovation Solution
The development of ligation-free methods for nucleic acid library preparation, which involve single-sided PCR, terminal transferase-mediated addition of nucleotides, and subsequent PCR to add adapters to the nucleic acids, allowing for the generation of nucleic acid libraries suitable for small and degraded samples without the need for ligation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If ligation-based techniques are used for library preparation, then adapters can be introduced onto nucleic acids, but the method is not suitable for small and highly degraded samples
Solution Approach 1:
The patent extracts and removes the ligation step from the conventional library preparation workflow. Instead of using ligation to attach adapters, the invention uses PCR-based methods where adapters are incorporated directly into primers, eliminating the need for separate ligation reactions and making the method suitable for degraded samples.
Solution Approach 2:
The patent replaces the mechanical/enzymatic ligation process with a PCR-based chemical amplification process. The adapter attachment is achieved through primer extension and PCR amplification rather than through ligase enzyme activity, fundamentally changing the mechanism of adapter incorporation.
2Productivity
If in vitro transcription is used to generate RNAs, then RNA can be produced, but untemplated nucleotides are added to the 3′ ends affecting function
Solution Approach 1:
The patent applies preliminary action by adding a tail sequence to the DNA template before in vitro transcription. This tail sequence serves as a template that directs the RNA polymerase to terminate at the correct position, preventing the addition of untemplated nucleotides and ensuring precise 3′ ends in the transcribed RNA.
Solution Approach 2:
The patent introduces a tail sequence as an intermediary element between the coding sequence and the transcription termination signal. This intermediary tail sequence mediates the transcription process by providing a defined template that controls where transcription stops, thereby preventing run-on transcripts with untemplated nucleotides.
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
These methods enable the efficient preparation of nucleic acid libraries from small and degraded samples, improving the recovery of genetic information and reducing the impact of untemplated nucleotides on RNA function.
Implementation Method 1
contacting the plurality of first single-sided PCR products with a terminal transferase under conditions sufficient to transfer dNTPs to the 3′ ends of the plurality of first single-sided PCR products
Implementation Method 2
contacting the sample of nucleic acids, a plurality of first polymerase chain reaction (PCR) primers and a polymerase under conditions that allow PCR to occur, thereby generating a plurality of first single-sided PCR products
Implementation Method 3
contacting the plurality of nucleic acids comprising 3′ tails with a plurality of first adapters and a reverse transcriptase under conditions sufficient for first strand complementary DNA (cDNA) synthesis to occur
Data Source
AI summary
Provided are compositions and methods of making a guide nucleic acids (gNAs), methods of using gNAs. and ligation free methods of preparing libraries of nucleic acids for downstream applications such as high-throughput sequencing.