DNA Fragment Detection via Cyclization for Low Sensitivity

Solution Overview

Problem

Current DNA fragment detection methods, particularly PCR amplification, face challenges with low sensitivity due to the requirement for complete DNA templates and are ineffective for fragmented DNA samples, such as those from plasma or FFPE tissues, which are often incomplete and difficult to amplify.

Innovation Solution

A DNA fragment detection method involving the design of primers that extend backwards, allowing for cyclization of DNA fragments, followed by PCR amplification, which increases the adaptability and effective template amount, enhancing detection sensitivity even with incomplete DNA fragments.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If traditional PCR amplification is used for DNA fragment detection, then the method is simple and fast, but the detection sensitivity is low due to requirement for complete DNA templates

Engineering Contradiction:

Improvedetection sensitivityVSAvoidadaptability to fragmented DNA

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The patent inverts the traditional linear amplification approach by implementing cyclization of DNA fragments first, then performing PCR amplification on the cyclized products. This inversion allows fragmented DNA to be converted into circular templates that can be effectively amplified even when fragments are incomplete, thereby improving detection sensitivity for fragmented DNA samples

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The patent changes the topological parameter of DNA from linear to circular through cyclization. This parameter change transforms the DNA structure into a form that is more suitable for PCR amplification, allowing the amplification process to work effectively with fragmented DNA templates that would otherwise be unsuitable for traditional linear PCR

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If PCR amplification requires complete DNA templates between primers, then the amplification specificity is high, but the detection sensitivity for fragmented DNA is low

Engineering Contradiction:

Improvedetection sensitivityVSAvoidamplification success rate

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent inverts the traditional linear amplification approach by implementing cyclization of DNA fragments first, then performing PCR amplification on the cyclized products. This inversion allows fragmented DNA to be converted into circular templates that can be effectively amplified even when fragments are incomplete, thereby improving detection sensitivity for fragmented DNA samples

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The patent creates multiple copies of the DNA fragment through PCR amplification after cyclization. By converting the fragmented DNA into circular templates and then amplifying them, the method generates sufficient copy numbers of the target sequence, enabling detection even when the original template is fragmented or present in low quantities

Inventive Principle:

Principle #26Copying

3Duration of action of stationary object

If FFPE method is used for tissue storage, then the tissue can be stored for long time, but the DNA becomes cross-linked and fragmented

Engineering Contradiction:

Improvestorage durationVSAvoidDNA integrity

Core Design Contradiction:

Duration of action of stationary objectVSStability of the object's composition

Solution Approach 1:

The patent converts the harmful effect of DNA fragmentation caused by FFPE processing into a beneficial feature. By designing a detection method that specifically targets and amplifies fragmented DNA through cyclization, the patent turns the previously problematic fragmented state into the expected and exploitable condition, allowing successful detection from FFPE samples

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Solution Approach 2:

The patent changes the topological parameter of DNA from linear to circular through cyclization. This parameter change transforms the DNA structure into a form that is more suitable for PCR amplification, allowing the amplification process to work effectively with fragmented DNA templates that would otherwise be unsuitable for traditional linear PCR

Inventive Principle:

Principle #35Parameter changes

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 significantly improves the detection sensitivity of DNA fragments by enabling amplification with incomplete templates, allowing for the detection of mutations and variations in DNA samples that were previously undetectable, including those from plasma and FFPE tissues.

Implementation Method 1

cyclization is CIRCLIGASE mediated double-chain DNA cyclization

Methodology Applied

Scientific EffectDNA cyclization: Enzyme

Data Source

PatentUS9540687B2DNA fragment detection method, DNA fragment detection kit and the use thereof

Publication Date: 2017.01.10 BERRYGENOMICS CO LTD

AI summary

The disclosure claims a cleaved Deoxyribonucleic acid (DNA) detection method, a DNA fragment detection kit and use thereof. Wherein, the method includes the steps of: designing primers according to a test site or a test region of the DNA fragment; cyclizing the DNA fragment to obtain acyclized DNA; implementing Polymerase Chain Reaction (PCR) amplification for the cyclized DNA by using the primers; and detecting the PCR amplification product. In the disclosure, by cyclizing the DNA fragment, the amplification can be implemented even if only one PCR primer can match with a template, thus, the adaption range and effective template amount of the primer amplification can be greatly increased, and the detection sensitivity of the DNA fragment can be greatly improved.