Selective Single-Stranded DNA Amplification for Low-Abundance cDNA

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Solution Overview

Problem

Current cDNA normalization methods, such as the DSN and hydroxyapatite column methods, suffer from depletion of low abundance sequences, bias against longer sequences, and high risk of PCR artifacts, making them unsuitable for efficient RNA sequencing.

Innovation Solution

A method involving denaturation and re-hybridization of cDNA strands followed by selective amplification using pre-attached adapters and oligonucleotide dimers to enhance low abundance cDNA without depletion, reducing PCR artifacts and bias.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If DSN or hydroxyapatite column normalization methods are used to reduce high abundance house-keeping genes, then sequencing coverage of low abundance genes is improved, but low abundance sequences are depleted and PCR artifacts increase

Engineering Contradiction:

Improvedetection sensitivity of low abundance genesVSAvoidaccuracy of sequence representation

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

Instead of depleting high abundance sequences through enzymatic digestion or column binding, the patent inverts the approach by selectively amplifying low abundance single-stranded cDNA molecules. This is achieved by exploiting the fact that low abundance sequences remain single-stranded after denaturation while high abundance sequences re-hybridize to form double-stranded molecules, which are then selectively amplified using ssDNA-specific polymerases and adapters.

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The patent changes the physical state parameter of cDNA molecules by controlling denaturation and re-hybridization conditions. By adjusting temperature and ionic strength parameters, the method creates conditions where low abundance sequences remain single-stranded while high abundance sequences form double-stranded structures, enabling selective amplification based on strand state rather than sequence abundance.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If cDNA normalization is performed to achieve uniform distribution of unique genes, then sampling efficiency is improved, but starting material requirements increase

Engineering Contradiction:

Improvesampling efficiency for gene detectionVSAvoidamount of starting cDNA required

Core Design Contradiction:

ProductivityVSQuantity of substance

Solution Approach 1:

The patent applies local quality by treating different cDNA populations differently based on their abundance characteristics. Low abundance single-stranded cDNA molecules are selectively targeted for amplification using ssDNA-specific adapters and polymerases, while high abundance double-stranded molecules are left unamplified or amplified at lower efficiency, creating a normalized distribution without requiring large amounts of starting material.

Inventive Principle:

Principle #3Local quality

3Measurement precision

If selective amplification of single stranded cDNA is performed using ssDNA-specific adapters and polymerases, then low abundance sequences are enriched, but risk of amplification bias is reduced

Engineering Contradiction:

Improvequantification accuracy of low abundance genesVSAvoidcomplexity of amplification protocol

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent introduces ssDNA-specific adapters as intermediary molecules that bridge the gap between single-stranded cDNA templates and amplification machinery. These adapters contain sequences that anneal to ssDNA and provide binding sites for ssDNA-specific polymerases, enabling selective amplification of low abundance sequences without requiring complex purification or separation steps.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 method requires less starting material, minimizes over-depletion, and reduces PCR artifacts, enabling effective detection of low abundance genes and sequences.

Implementation Method 1

denaturing the cDNA sample to produce single stranded cDNA templates

Methodology Applied

Scientific EffectDenaturation:

Implementation Method 2

re-associating the cDNA sample to produce a mixture of post-association single stranded cDNA templates and post-association double stranded cDNA templates

Methodology Applied

Scientific EffectRe-hybridization:

Implementation Method 3

annealing a 5' adapter complex to the 5' pre-attached adapter of at least one post-association single stranded cDNA template

Methodology Applied

Scientific EffectAnnealing: Annealing

Implementation Method 4

ligating an oligonucleotide from the 5' adapter complex to the 5' pre-attached adapter of the post-association single stranded cDNA template

Methodology Applied

Scientific EffectLigation:

Implementation Method 5

selectively amplifying the cDNA sample using primers specific to the ligated oligonucleotides

Methodology Applied

Scientific EffectPCR amplification:

Data Source

PatentUS20250297304A1Amplification of Single Stranded DNA

Publication Date: 2025.09.25 WOBBLE GENOMICS LTD

AI summary

The present invention relates to methods, kits and compositions for selective amplification of single stranded DNA. The invention is useful in generating a normalized cDNA fraction and it can be used in various RNA and DNA sequencing applications to amplify DNA templates having pre-attached adapters. We describe a method of selective amplification of single stranded cDNA. We also describe an oligonucleotide dimer composition for use in a method and a selective amplification kit for selectively amplifying low abundance cDNA from a cDNA sample.