Blocking Oligonucleotides for RNA Library Purity

Solution Overview

Problem

The existing methods for preparing RNA sequencing libraries are hindered by the formation of unwanted adapter:adapter by-products during ligation reactions, which require cumbersome gel purification steps, limiting the yield and automating potential of RNA sequencing processes.

Innovation Solution

A single-stranded blocking oligonucleotide is designed to specifically bind to the adapter:adapter by-products, preventing their amplification by interfering with reverse transcription, thus eliminating the need for gel purification and simplifying the library preparation process.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If gel purification steps are used to remove adapter:adapter by-products, then the purity of RNA sequencing libraries is improved, but the complexity of the library preparation process increases and productivity decreases

Engineering Contradiction:

Improvepurity of RNA sequencing libraryVSAvoidcomplexity of library preparation process

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent extracts and removes the harmful adapter:adapter by-products from the ligation reaction mixture through gel purification, separating them from the desired RNA:adapter products. This extraction approach directly addresses the purity issue while maintaining the overall library preparation workflow.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent performs preliminary purification of the ligation reaction products before proceeding to subsequent library preparation steps. By removing by-products early in the process, the patent prevents them from interfering with later amplification and sequencing steps, thereby improving final library quality without requiring multiple purification stages.

Inventive Principle:

Principle #10Preliminary action

2Productivity

If gel purification steps are used to remove adapter:adapter by-products, then the yield of RNA sequencing libraries is improved, but the time required for library preparation increases

Engineering Contradiction:

Improveyield of RNA sequencing libraryVSAvoidtime required for library preparation

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The patent extracts and removes the harmful adapter:adapter by-products from the ligation reaction mixture through gel purification, separating them from the desired RNA:adapter products. This extraction approach directly addresses the purity issue while maintaining the overall library preparation workflow.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent performs preliminary purification of the ligation reaction products before proceeding to subsequent library preparation steps. By removing by-products early in the process, the patent prevents them from interfering with later amplification and sequencing steps, thereby improving final library quality without requiring multiple purification stages.

Inventive Principle:

Principle #10Preliminary action

3Device complexity

If adapter:adapter by-products are not removed, then the library preparation process is simplified, but the amplification efficiency decreases due to preferential amplification of by-products

Engineering Contradiction:

Improvesimplicity of library preparation processVSAvoidamplification efficiency

Core Design Contradiction:

Device complexityVSReliability

Solution Approach 1:

The patent extracts and removes the harmful adapter:adapter by-products from the ligation reaction mixture through gel purification, separating them from the desired RNA:adapter products. This extraction approach directly addresses the purity issue while maintaining the overall library preparation workflow.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent performs preliminary purification of the ligation reaction products before proceeding to subsequent library preparation steps. By removing by-products early in the process, the patent prevents them from interfering with later amplification and sequencing steps, thereby improving final library quality without requiring multiple purification stages.

Inventive Principle:

Principle #10Preliminary action

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

The use of blocking oligonucleotides effectively inhibits the amplification of by-products, enhancing the yield of RNA sequencing libraries and enabling a more automated and streamlined RNA library preparation process.

Implementation Method 1

a first segment hybridizes with a first nucleotide adapter molecule and the second segment hybridizes with a second nucleotide adapter molecule

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentUS11136616B2Oligonucleotides and methods for the preparation of RNA libraries

Publication Date: 2021.10.05 LIFE TECHNOLOGIES CORP

AI summary

Disclosed are compositions and methods for the preparation of RNA libraries for sequencing, gene expression profiling, microarray and other uses and for simplification of the library preparation process. The disclosure provides blocking oligonucleotides which bind to byproduct nucleic acid molecules formed during the ligation of adapters to nucleic acid segments prior to sequencing and inhibit or block amplification of the byproduct nucleic acid molecules in subsequent amplification reactions. Methods for library preparation using blocking oligonucleotides are also provided.